Potent 5-Cyano-6-phenyl-pyrimidin-Based Derivatives Targeting DCN1-UBE2M Interaction

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Potent 5‑Cyano-6-phenyl-pyrimidin-Based Derivatives Targeting

DCN1−UBE2M Interaction

Wenjuan Zhou,^†,‡,∥Liying Ma,^†,∥Lina Ding,^†Qian Guo,^†Zhangxu He,^†Jing Yang,^†Hui Qiao,^†

Lingyu Li,^†Jie Yang,^†Shimin Yu,^†Lili Zhao,^†Shaomeng Wang,^†,§Hong-Min Liu,*^,†Zhenhe Suo,*^,‡

and Wen Zhao*^,†

^†State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key Laboratory of Advanced Pharmaceutical Technology,

Ministry of Education of China; School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan

450001, China

^‡Department of Pathology, Oslo University Hospital; Faculty of Medicine, University of Oslo, Oslo 0379, Norway

^§The Rogel Cancer Center and Departments of Internal Medicine, Pharmacology, Medicinal Chemistry and Pathology, University of

Michigan Medical School, Ann Arbor, Michigan 48109, United States

S

* Supporting Information

ABSTRACT: Neddylation of the Cullin-RING E3 ligases (CRLs) regulates the homeostasis of approximately 20% of cellular

proteins. Defective in cullin neddylation 1 (DCN1), as a co-E3 ligase, interacts with UBE2M to enhance the activation of CRLs,

and this interaction is emerging as a therapeutic target for human diseases. Here, we present a series of pyrimidin-based small

molecular inhibitors targeting DCN1−UBE2M interaction. After ﬁnding a novel inhibitor DC-1 with IC₅₀= 1.2 μM, we

performed a series of chemical optimizations, which ﬁnally led to the discovery of a potent thiazole containing 5-cyano-6-

phenylpyrimidin-based inhibitor DC-2 (IC₅₀= 15 nM). Next, using protein and cellular thermal shift assays,

coimmunoprecipitation, molecular docking, and site-speciﬁc mutation experiments, we further proved that DC-2 speciﬁcally

inhibited the interaction of UBE2M and DCN1 at molecule and cellular levels, resulting in the decrease of cullin3 neddylation

and accumulation of its substrate, NRF2. Our ﬁndings indicate that DC-2 may serve as a novel lead compound for speciﬁc

derivatives targeting DCN1−UBE2M interaction.

neddylation has been recognized to be highly activated in

various cancers,^20,27−33which draws much attraction for

oncologic drug discovery, especially on the ﬁnding of

NEDD8 E1 inhibitor Pevonedistat (MLN4924).^10,23−26

MLN4924 covalently binds to NEDD8 E1, blocking all

CRLs neddylation,⁷thus causing accumulation of CRL

substrates.^7,10Because MLN4924 possesses immense anti-

cancer eﬀects both in vitro and in vivo,^10,27−43it has been

approved for Phase II clinical trials for treatment of human

acute myeloid leukemia, nonsmall cell lung cancer, and

mesothelioma.²⁹However, as a result of its broad ablation of

neddylation, MLN4924 has a series of toxicities. Therefore,

INTRODUCTION

■

The ubiquitin−proteasome system (UPS) is integral to

maintaining cellular protein homeostasis by regulating

degradation of intracellular proteins.¹⁻⁷The ubiquitylation

pathway is executed by the coordinated eﬀorts of the E1

(ubiquitin-activating enzyme), E2 (ubiquitin-conjugating

enzyme), and E3 (ubiquitin ligase enzyme) proteins.⁵Like

ubiquitination,⁸neddylation is a novel type of posttranslational

modiﬁcation, in which the ubiquitin-like molecule NEDD8 is

added to its target proteins and thus regulates their

functions.^9,10Neddylation also consists of a tripartite

enzymatic cascade, including the NEDD8-activating enzyme

E1 (NAE, APPBP1 (NAE1), and UBA3 heterodimer), two

NEDD8-conjugating enzymes E2s (UBE2F and UBE2M, also

known as UBC12), and NEDD8-E3 ligases,^9,10with cullins

being the best-characterized substrates.¹¹⁻²²Up to now,

Received: January 1, 2019

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Figure 1. Representative examples of DCN1 inhibitors.

alternative targeting speciﬁc CRLs may have more potential

and be safer for cancer treatment.³⁰⁻³²

neddylation and the accumulation of NRF2 and NRF2′

downstream proteins, HO-1 and NQO1. Our overall ﬁndings

indicate that DC-2 may serve as a novel lead compound for

speciﬁc derivatives targeting the DCN1−UBE2M interaction.

DCN1 (defective in cullin neddylation 1), also called

DCUN1D1, DCNL1, or SCCRO, is a highly conserved gene

and ampliﬁed along the 3q26.3 in most squamous cell

carcinomas and some other human cancers.³³⁻³⁹As a co-E3

ligase, it binds to the activation complex of Cullin-RBX1-

UBE2M-NEDD8 to increase neddylation eﬃciency.^16,40−42

Researchers have found that blocking UBE2M−DCN1

interaction can reduce cullin3 neddylation.^30,32,43Moreover,

DCN1 overexpression has been reported to be associated with

poor survival outcome, and DCN1 depression signiﬁcantly

reduces cancer cell growth and invasive ca-

pacity.^{10,33−35,42,44−51}Therefore, DCN1 is considered as a

promising and attractive anticancer target.

From the X-ray crystallographic structure of DCN1 with

UBE2M, researchers have conﬁrmed that during neddylation,

the N-terminal acetylated UBE2M can dock into the

hydrophobic pocket of DCN1, and this pocket could be

used for designing DCN1 inhibitors.^21,52Up to now, four

papers have reported the discovery of DCN1−UBE2M

interaction inhibitors,^30−32,43also known as DCN1 inhibitor.

NAcM-COV, the ﬁrst discovered inhibitor, showed highly

speciﬁc eﬀects on blocking the N-terminal acetylation-

dependent interaction of UBE2M with DCN1, resulting in

the reduction of CUL1 and CUL3 neddylation in a squamous

lung cancer cell line (HCC95).^31,43At the same time, Zhou et

al, discovered two high-aﬃnity inhibitors DI-591 and DI-404,

which can selectively inhibit the neddylation of cullin 3 over

other cullins, leading to the accumulation of cullin 3′s

substrate, NRF2 (Figure 1).^30,32In this study, we screened

our in-house structurally diverse molecular library (ca. 1000

compounds) and ﬁnally found a novel and potent pyrimidine-

based DCN1−UBE2M interaction mediator DC-1, using both

ﬂuorescence polarization (FP) and homogeneous time-

resolved ﬂuorescence (HTRF)⁵³assays. After extensive

structure−activity relationship (SAR) eﬀorts, a potent thiazole

containing 5-cyano-6-phenylpyrimidin-based inhibitor DC-2

was discovered (Figure 2). DC-2 speciﬁcally targets DCN1−

UBE2M interaction, leading to the inhibition of cullin3

RESULTS AND DISCUSSION

■

Chemistry. The general synthesis route of the target

pyrimidine-thiourea hybrids is depicted in Scheme 1.

Benzaldehydes 1a−m, ethyl cyanoacetate 2, and thiourea 3

were prolonged heated in ethanol containing potassium

carbonate to obtain 6-aryl-5-cyano-2-thiouracils 4a−m. Com-

pounds 4a−m then reacted with the 3-bromoprop-1-yne, 3-

bromoprop-1-ene, 1-bromopropane, and phosphorus oxy-

chloride in dioxane to obtain the target derivatives 5a−o.⁵⁴

Compound 53 was prepared via click reaction of compound 5a

with benzyl azide. After that, these highly activated

intermediates (5a−o and 53) were reacted with appropriate

mercapto heterocyclics and anilines to obtain compounds 6−

52 and 54.

Development of FP- and HTRF-Based Competitive

Binding Assays. To acquire stable and reliable binding

aﬃnities of our compounds in vitro, two methods (FP and

HTRF) were used for screening DCN1−UBE2M interaction

inhibitors. On the basis of the principle that the N-terminally

acetylated UBE2M can interact with DCN1,⁵⁵UBE2M^NAc1−12

derivative ﬂuorescently labeled tracer (FAM-782) and His-

tagged DCN1 recombinant protein were used to develope the

ﬂuorescence polarization (FP) assay.³⁰In addition, human

GST-tagged DCN1 recombinant protein (Figure S1A) and

AcUBE2M¹⁻²¹-biotin peptides were applied to establish the

HTRF screening system. The detailed schematic diagrams are

shown in Figure 3A and B.

Biochemical Activity of the Candidate Compounds

against DCN1−UBE2M Interaction and the Structure−

Activity Relationship (SAR) Studies. All of the compounds

synthesized in this study were examined for their inhibitory

eﬀects on DCN1−UBE2M interaction in vitro by both FP and

HTRF assays.³⁰Compounds DI-591 and NAcM-COV were

chosen as positive controls for FP³⁰and HTRF assays,⁴³

respectively. All IC₅₀values reported in this study were

obtained from at least three independent experiments. The

results are summarized in Tables 1−4.

To investigate the eﬀect of the coumarin group on inhibitory

activity, compounds 7−10 were synthesized initially, and their

inhibition activities are shown in Table 1. As compared to the

lead compound DC-1, compounds 7−9 with diﬀerent

heterocyclic substitution at R₁caused a dramatic loss of

activity. However, compound 10 with aromatic ring sub-

stitution performed with a more potent inhibitory eﬀect. These

ﬁndings indicate that the R₁site plays a vital role in inﬂuencing

their activities and the aromatic ring substitutions at R₁may be

Figure 2. Discovery of potent thiazole contained pyrimidin-based

inhibitor DC-2.

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a

Scheme 1. Synthesis of the Target Pyrimidine-Based Derivatives

a

Reagents and conditions: (a) absolute ethanol, absolute K₂CO₃, reﬂux, 10 h, 70−90%; (b) (i) 3-bromoprop-1-yne, 3-bromoprop-1-ene, 1-

bromopropane, dioxane, reﬂux; (ii) phosphorous oxychloride, reﬂux, 1 h, 50−80% (two steps); (c) benzyl azide, CuSO₄·5H₂O, sodium ascorbate,

THF−H₂O (1:1), rt, 80%; (d) appropriate mercapto heterocyclic and aniline, absolute ethanol, reﬂux, 6 h, 70−95%.

Figure 3. Detailed schematic diagrams for the principle of screening DCN1−UBE2M interaction inhibitors. (A) FP assay for DCN1 binding to

UBE2M^NAc1−12derivative. The FP value will be decreased after adding the compound, which can inhibit the DCN1−UBE2M interaction. (B)

HTRF assay for DCN1 binding to UBE2M^NAc1−21. Inhibitors will receive a low signal.

more important contributors in determining activity, as

compared to those with coumarin group substitution.

Next, through further structure−activity relationship (SAR)

studies, we found that the heteroatoms and diﬀerent

heterocyclic substitutions at R₁were also important for the

inhibitory activity (Table 3). Replacing the sulfur atom at R₂of

compound 10 with nitrogen atom (19) led to a complete loss

of activity. A similar trend was observed by changing the

tetrazole-thione to other amino analogues (20−25). In

contrast, sulfhydryl heterocyclic substitutions attached to the

4-position of the pyrimidine skeleton were well tolerated. As

compared to 10, some of these compounds showed

comparably or marginally increased potency, of which 34

with thiazolethiol group exhibited the most potent activity as

compared to other heterocyclic substitutions (26−33). The

extension of the side chain on nitrogen atom (26) has no

signiﬁcant eﬀect on activity, as compared to the corresponding

compound 10. In addition, we found that the number of

nitrogen atoms on the mercapto heterocycles was important

for the inhibitory activity: the tetrazole derivative 10 was more

potent than the pyrazole derivative 27, and the triazole (28),

pyridine (29) derivative without any detectable activity.

On the basis of the above ﬁndings, further modiﬁcations

were next focused on the substitution pattern and electronic

eﬀect on the aromatic ring of compound 10. As shown in

Table 2, no matter an electron-withdrawing or -donating group

substitution at R₁, the acquired compounds 11−17 display

increased inhibitory activity as compared to no substitution

compound 18. Compounds 11−14 with electron-withdrawing

groups at R1 have a more potent inhibitory eﬀect as compared

to compounds 15−17 with electron-donating groups (except

10). The substitution of multiple functional groups on

aromatic rings is beneﬁcial to the improvement of activity

(11 vs 12, 18 vs 17). Particularly, compound 14 with p-Br

substitution eﬀectively blocked the DCN1−UBE2M inter-

action with an IC₅₀value of 65 nM, more potent than 12 with

p-Cl substitution. These ﬁndings suggest that the number,

type, and position of substituent and electronic eﬀect on the

aromatic ring have signiﬁcant eﬀects on the activity.

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Table 1. Optimizing the Coumarin Group in the Structure of Compound 6 (DC-1) (Compounds 6−10)

a

IC₅₀values were obtained from three independent repeats and represented as mean SD. ND means the value was not determined.

Particularly, replacement of the nitrogen (N) atom in 28 with

the sulfur (S) atom yielded 30, which signiﬁcantly increased

the inhibitory activity with an IC₅₀value of 195 nM. A similar

trend was also observed in compounds 31−34, which

suggested that the sulfur atom plays an important role in

inhibitory activity. In addition, the introduction of hydrophilic

functional groups (32, 33) reduced the inhibitory activity to

some extent. Interestingly, as compared to 34, it was found

that derivatizations of the oleﬁnic bond on the thiazolethiol

group (35−37) exhibited disappearance of activity, which

indicated that the increase of steric hindrance at R₂was

detrimental to the activity.

To further investigate the importance of the two sites at R₁

and R₂of 34, we simultaneously changed them and

synthesized compounds 38−50. As compared to 34, some of

these compounds showed comparable potency against DCN1,

of which 42 (also named as DC-2) showed the most potent

inhibitory eﬀect on the DCN1−UBE2M interaction with an

IC₅₀value of 26 nM. In addition, compound 39 with chlorine

substitution in the para position at the phenyl ring in R₁

displayed more potent inhibitory activity as compared to the

meta- and disubstituted compounds (38 and 41). A similar

trend was also observed (46 vs 47, 48), which suggested that

the position of substituent at the phenyl ring in R1 plays an

important role in the inhibitory activity. Furthermore,

replacement of the phenyl group at R₁with aromatic

heterocycle yielded 43−45, which signiﬁcantly decreased the

inhibitory activity, as well as 46−49 versus 50, that further

suggested that the position of substituent and electronic eﬀect

at R₁were important for the activity. Moreover, the

importance of terminal alkyne moiety was also evaluated.

Because of the change of the propargyl group to ethene or

ethyl group, compounds 51 and 52 exhibited no detectable

(>10 000 nM) DCN1−UBE2M inhibitory eﬀect as compared

to 39, which may be related to the hydrophobic interactions by

forming π−π stacking with DCN1−UBE2M. In addition,

replacing the propargyl group by triazole via click chemistry

caused a dramatic loss of activity (54 vs 39), which indicated

that the increase of steric hindrance at R₃was detrimental to

the activity. These modiﬁcations and SAR studies reveal that

the terminal alkyne group is also critical for their inhibitory

activity, and the introduction of hydrophobic functional groups

of appropriate size at R₃may be beneﬁcial to the improvement

of activity.

In summary, starting from a potent pyrimidine-based

DCN1−UBE2M interaction mediator DC-1 (compound 6)

in our in-house structurally diverse molecular library (ca. 1000

compounds), DC-2 (compound 42) containing 5-cyano-6-

phenyl-pyrimidin was ﬁnally obtained with MW of 380, and

two IC₅₀values of 26.89 0.24 (K_i= 20.83 0.18 nM) and

15.71 1.19 nM (K_i= 13.66 1.03 nM) from the FP and

HTRF assays (Figure 4A and 4B), respectively, resulting in

about 80-fold improved potency. Furthermore, DC-2 has

aqueous solubility in phosphate buﬀer (PBS, pH 7.4) to some

extent (Figure S2). Therefore, DC-2 was chosen for the

following target activity evaluation experiments. The com-

pound 35 (DC-2N), which had a structure similar to DC-2 but

presented no obvious inhibitory eﬀect on DCN1−UBE2M

interaction (IC₅₀> 10 μM), was chosen as a negative control.

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Table 2. Optimization of the Substitution Pattern and Electronic Eﬀect on the Aromatic Ring of Compound 10 (Compounds

11−18)

a

IC₅₀values were obtained from three independent repeats and represented as mean SD. ND means the value was not determined.

DC-2 Binds to DCN1 and Increases Its Stability in

Vitro. Next, the protein thermal shift assay was conducted to

assess whether DC-2 can directly bind to DCN1 and adjust its

stability in vitro.^56,57As shown in Figure 5, as compared to

DMSO control and DC-2N, DC-2 obviously caused an about

7 °C increase of the DCN1 melting temperature, similar to the

eﬀects of the positive control NAcM-COV. These ﬁndings

indicate that DC-2 can directly bind to DCN1 and thus raise

its thermal stability in vitro.

Binding Aﬃnities of DC-2 on DCN Proteins. Previous

studies have reported that there are ﬁve DCN1 homologues

(DCNs: 1−5) in humans,^33,50which contain a highly conserved

central and C-terminal PONY domain, but possess distinct N-

terminal regions.^33,50,58,59The DCN1−5 PONY domains can

interact with cullins to stimulate neddylation with diﬀerent

eﬃciencies.^55,60Therefore, we further measured the binding

aﬃnities of DC-2 on DCN1−5 through competitive binding

assay. As shown in Table 5, DC-2 also has a high binding

aﬃnity on DCN2, whose PONY domain in human shares 82%

identity to that of DCN1.⁴³However, it shows relatively weak

binding eﬀects (K_i> 500 nM) on DCN3−5, as compared to

that of DCN1. These data suggest that DC-2 has speciﬁcity

and selectivity for DCN1 and 2, similar to the previously

reported inhibitors (DI-591, NAcM-COV, and DI-

404).^30,32,43

Furthermore, because the N-terminal domain of UBE2M

has interaction with both DCN1 and NEDD8 E1 subunit

UBA3,^52,61we intended to determine whether DC-2 also has

inhibitory eﬀects on E1’s activity and UBE2M neddylation. As

shown in Figure 6A and B, in the cell-free NAE activity assay,

even though MLN4924 signiﬁcantly inhibits UBE2M

neddylation, compound DC-2 exhibits no obvious eﬀect on

NEDD8∼UBE2M at concentration up to 100 μM, indicating

that compound DC-2 has no eﬀect on E1’s activity through

ATP initiation and UBE2M neddylation.

Molecular Docking of DC-2 and DCN1. To investigate

the binding mode of compound DC-2 with human DCN1,

molecular docking study adopting the software MOE (The

Molecular Operating Environment) version 2015.10 was

carried out. Because the X-ray structure (PBD ID: 3TDU⁵²)

obtained from the RSCB Protein Data Bank is the crystal

structure of DCN1 in a complex with the N-terminal-

acetylated UBE2M peptides and cullin 1, and our inhibitors

were found on the basis of the principle that inhibitors

compete with the N-terminal-acetylated UBE2M peptides to

interact with DCN1, 3TDU was thus selected as the receptor

to acquire the binding mode of DC-2 with DCN1. As shown in

Figure 7A and B, compound DC-2 can be successfully docked

into the active site of DCN1. There are 12 of 20 top-score

docking conformations, which adopt a similar docking pose.

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Table 3. Optimization of the Heteroatoms and Diﬀerent Heterocyclic Substitutions of Compound 10 (Compounds 19−37)

a

IC₅₀values were obtained from three independent repeats and represented as mean SD. ND means the value was not determined.

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Table 4. Optimization of the Aromatic and Heterocyclic Substitutions at R₁, R₂, and R₃of Compound 34 (Compounds 38−54)

a

IC₅₀values were obtained from three independent repeats and represented as mean SD. ND means the value was not determined.

Among them, the conformation with the highest score is

illustrated in Figure 7A. The nitrogen atom of cyan group of

DC-2 forms a hydrogen bond with the hydroxyl group of the

side chain of Tyr181 with a distance of 2.09 Å. The sulfur atom

connecting the thiazole ring of DC-2 forms a nontraditional

hydrogen bond with the carbonyl group of the backbone of

Pro97 with a distance of 3.55 Å. Besides, the methylbenzene

group of DC-2 forms arene-H interaction with Phe164. The

binding pocket surface of DCN1 and compound DC-2 is

shown in Figure 7B. The methylbenzene group of DC-2 is

buried into the inside hydrophobic pocket surrounded by

Phe117, Phe89, Ile86, Ala106, Val102, and Leu103. The

propargyl group of DC-2 is located in the hydrophobic regions,

which are formed by Gln87, Ile83, Ile86, and Cys115. In

addition, the thiazole ring of DC-2 forms hydrophobic

interactions with Tyr181, Leu184, and Met177. All of these

computer-based predicted interactions indicate that compound

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Figure 4. Compound DC-2 (compound 42) combines with DCN1 and thus inhibits UBE2M−DCN1 interaction in vitro. Compound DC-2 was

added into the screening systems, and its inhibition rates (%) at diﬀerent concentration were determined by both FP (A) and HTRF (B) assays,

respectively. Data are presented as means SD. Three individual experiments were performed for each group.

F164S and Y181I mutations, indicating that there is no

interaction between the DCN1 mutations and AcUBE2M¹⁻²¹

-

biotin peptides. However, these parameters in DCN1 P97T

mutation remained almost the same as those in wild-type

DCN1, indicating that the P97T mutation did not aﬀect its

binding aﬃnity with AcUBE2M¹⁻²¹-biotin (Figure 7C and D).

To conﬁrm this discovery, we further did the label-free

BioLayer Interferometry (BLI) assay. Consistent with our

above ﬁndings, DCN1 F164S and Y181I mutations lost the

binding aﬃnity with AcUBE2M¹⁻²¹-biotin peptide rather than

DCN1 P97T mutation (Figure S3). All of these ﬁndings

suggest that both Phe164 and Tyr181 residues are crucial for

DCN1−UBE2M interaction, and the inhibition eﬀect of DC-2

may be related to its binding interaction with these two

residues.

Figure 5. Compound DC-2 increases the stability of DCN1 in vitro.

Normalized thermal shift assay response for recombinant human

DCN1 in the presence of 0.5 mM DC-2 (orange) and DC-2N

(green). NAcM-COV (red) was used as a positive control. DMSO

(blue) was used as a blank control. Three individual experiments were

performed for each group.

DCN1 Was Highly Activated in Several Human

Cancer Cell Lines. To choose suitable cancer cell lines for

the further experiments, the DCN1 protein levels in several

human cancer cell lines, including lung cancer cell lines (PC9,

A549, and H1975), esophageal cancer cell lines (EC109,

EC9706, KYSE70, KYSE140, and TE-1), liver cancer cell lines

(SMMC-7721, BEL-7402, and ZIP177), prostatic cancer cell

line (PC3), and breast cancer cell line (MCF7), were

measured. We found that DCN1 was highly expressed in

these cancer cell lines, as compared to those in normal cell

lines: GES-1, Het-1A, and L02, respectively (Figure 8A−C).

Interaction of DC-2 with DCN1 at the Cellular Level.

To further determine whether DC-2 can target intracellular

DCN1, cellular thermal shift assay (CETSA) was performed in

one of the DCN1 overexpressed cancer cell line H1975. As

shown in Figure 9A and D, DCN1 protein started to degrade

at 53 °C in DMSO or 10 μM DC-2N treated cells (Figure

9A,B,D, and E), while it was very stable from 43 to 61 °C in

the 10 μM DC-2 treated cells, similar to that treated by

positive compound DI-591 (Figure 9A and D).³⁰In addition,

DC-2 increases DCN1 thermal ability at a concentration low

to 1 μM at 55 °C (Figure 9B and E). These ﬁndings indicate

that DC-2 can engage the cellular DCN1 protein and increase

its thermal ability. Furthermore, the DCN1 level, which was

pulled down by UBE2M antibody, was obviously decreased in

DC-2 treated cells, as compared to those in DMSO or DC-2N

treated groups (Figure 9C and F). These ﬁndings further

DC-2 could be well and speciﬁcally docked into the binding

pocket of DCN1.

Site-Speciﬁc Mutations in DCN1 Binding Pockets

with DC-2. Previous studies have reported that the Pro97,

Phe164, and Tyr181 residues of human DCN1 are involved in

the interaction of DCN1 and UBE2M, especially Tyr181,

which can clamp the UBE2M’s N-acetyl-Met1 and Ile2,

pressing UBE2M’s N-acetyl-Met into DCN1’s hydrophobic

pocket.^52,55In addition, the reported DCN1−UBE2M

interaction inhibitors all have interactions with these three

residues, either by forming hydrogen bonds or by ﬁtting into

the hydrophobic pocket.^30,32,43Therefore, we hypothesize that

these three residues may play an important role in DCN1−

UBE2M interaction. Furthermore, our above docking results

also have conﬁrmed that DC-2 has relatively strong

interactions with Pro97, Phe164, and Tyr181 residues of

DCN1 (Figure 7A and B). Therefore, we speculate that the

inhibitory eﬀects of compound DC-2 on DCN1−UBE2M

interaction may be also related to these three amino acid

residues. To prove our hypothesis, the three residues of DCN1

(Pro97, Phe164, and Tyr181) were chosen to do the site-

speciﬁc mutations to Thr97 (P97T), Ser164 (F164S), and

Ile181 (Y181I), whose physicochemical properties are totally

diﬀerent from their original amino acids, respectively (Figure

S1C). In our in vitro FP and HTRF assays, we found the

complete loss of mp values and 665/615 ratios in DCN1

Table 5. Binding Aﬃnities of Compound DC-2 on the Indicated DCN1-5 Recombinant Proteins

a

DCN1 K_i(nM)

DCN2 K_i(nM)

DCN3 K_i(nM)

DCN4 K_i(nM)

DCN5 K_i(μM)

DC-2

13.66 1.03

66.19 1.72

591.72 5.17

807.01 9.42

2.14 0.32

a

K_ivalues were obtained from three independent repeats and represented as mean SD. ND means the value was not determined.

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Figure 6. Compound DC-2 exhibits no obvious inhibitory eﬀect on UBE2M neddylation level in vitro. (A) After adding 10 and 20 μM MLN4924

or 1, 10, and 100 μM DC-2 into the NEDD8, NEDD8 E1, and UBE2M complex, respectively, the NEDD8∼UBE2M formation was initiated by

ATP. (B) Densitometry shows relative protein expression level, which was analyzed by fold of DMSO with ATP group. Data are presented as

means SD. Three individual experiments were performed for each group.

Figure 7. Predicted binding mode of compound DC-2 in DCN1 binding pocket (PDB ID: 3TDU). (A) Residues forming interactions in the

docked complex. Compound DC-2 is shown as cyan stick; the residues associated with the compound are shown as white lines. Hydrogen bonds

are shown as magenta dash lines, and the arene−H interaction is shown as a red dash line. The corresponding distances are given in angstroms. (B)

The binding pocket surface of DCN1 and compound DC-2. Green area represents hydrophobic region, and red area represents exposed solvent

region. The mp values (C) and 665/615 ratios (D) were determined after adding increased concentration of recombinant wild-type (WT) DCN1

or its site-speciﬁc mutations: P97T, F164S, and Y181I. Data are presented as means SD. At least three individual experiments were performed for

each group.

suggest that compound DC-2 can bind to DCN1 and inhibit

the association of UBE2M and DCN1 at the cellular level.

Selectively Blocking the Neddylation of Cullin3 by

DC-2 in Lung Cancer Cells. Human DCN1 acts as a co-E3

ligase, which can interact with cullins and Rbx1 to stimulate

the neddylation of cullins in vivo, especially cullin1−

4.^{21,40,52,55,60,62}To examine the eﬀect of DC-2 on the

neddylation of cullins at cellular level, two DCN1 highly

expressed lung cancer cell lines H1975 and PC9 were treated

with compound DC-2 for 24 h, using DC-2N or MLN4924 as

a negative or positive control, respectively. We found that DC-

2 treatment resulted in a signiﬁcantly decreased cullin3

(CUL3) neddylation level, without obvious impact on the

neddylation of other cullins (CUL1, CUL2, CUL4A, CUL4B,

and CUL5, Figure 10A) in both cell lines. In addition, because

the inhibition of the cullins neddylation has been associated

with the accumulation of its substrates, the expression levels of

nuclear factor-erythroid 2 related factor 2(NRF2) (one

substrate of cullin3),⁶³⁻⁶⁵as well as NRF2’s downstream

proteins (NADPH: quinone oxidoreductase-1, NQO1) and

(heme oxygenase-1, HO-1), were then determined after DC-2

treatment.⁶⁶⁻⁶⁸We found that the expression levels of NRF2,

HO-1, and NQO1 were obviously increased in both H1975

and PC9 cells, similar to the ﬁndings treated by MLN4924

(MLN, Figure 10C). Yet the expression levels of CylinE1, p21

(two substrates of cullin1), and CDT1 (a substrate of

cullin4A) were not changed after DC-2 treatment (Figure

10C).⁶⁶⁻⁶⁸Furthermore, the inhibition eﬀects on the

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Figure 8. Expression levels of DCN1 in several human cancer cell lines. (A) Lung cancer cell lines: PC9, A549, and H1975. (B) Esophageal cancer

cell lines: EC109, EC9706, KYSE70, KYSE140, and TE-1. (C) Liver cancer cell lines: SMMC-7721, BEL-7402, and ZIP177. Prostate cancer cell

line: PC3. Breast cancer cell line: MCF7. Data are presented as means SD. At least three individual experiments were performed for each group.

*P < 0.05, **P < 0.01, ***P < 0.001 as compared to the normal control cell lines: GES-1, Het-1A, or L02, respectively.

Figure 9. Compound DC-2 speciﬁcally binds to DCN1 and blocks the interaction of DCN1−UBE2M at a cellular level. (A and B) H1975 cells

were treated with 10 μM compound DC-2, DI-591, or DC-2N for 5 h, respectively. They then were collected and heated from 43 to 61 °C (A).

H1975 cells were treated with 0, 0.3, 1, 3, 10 μM DC-2 or 10 μM DC-2N before being heated at 55 °C. Their DCN1 expression levels then were

determined by Western Blot (B). (C) Compound DC-2, but not DC-2N, blocks the association of DCN1 and UBE2M at the cellular level. After

H1975 cells were treated with compound DC-2N, DC-2, or NAcM-COV, the protein levels of UBE2M and DCN1, which were pulled down by

UBE2M antibody, were determined by Western Blot (right panel). The basal levels of DCN1 and UBE2M in cell lysates were determined by

Western Blot (left panel). (D−F) The bands intensities of proteins in (A)−(C), respectively. At least three individual experiments were performed

for each group.

neddylation of cullin3 (Figure 10B, E, G, and H) as well as the

accumulation of NRF2, NQO1, and HO-1 (Figure 10D, F, G,

and H) exhibited in a dose (0.3, 1, 3, 10, and 20 μM) and

time-dependent manner (0.5, 1, 3, 12, 24, and 48 h), without

signiﬁcant eﬀect on the neddylation of other cullins (CUL1−

5) (Figure 10B) and their conresponding substrates: CylinE1,

p21, and CDT1 (Figure 10D and G) in both H1975 and PC9

cells. These ﬁndings indicate the speciﬁcally inhibitory eﬀects

of DC-2 on the neddylation of cullin3 at the cellular level,

without signiﬁcant eﬀects on other cullins neddylation.

Moreover, silencing DCN1 by three small interfering RNA

(siRNA) in both PC9 and H1975 cells also decreased the

neddylation of cullin3 and caused the accumulation of NRF2,

NQO1, and HO-1 (Figure 11A−C). Furthermore, DC-2

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Figure 10. Compound DC-2 inhibits intracellular cullin3’s neddylation process and subsequently increases its substrate NRF2 as well as NRF2′

downstream proteins: HO-1and NQO1 with time (G and H) and dose (A−F)-dependent manner. Densitometry shows the relative protein

expression level normalized by GAPDH. Data are presented as means SD. Three individual experiments were performed for each group. *P <

0.05, **P < 0.01, ***P < 0.001 as compared to the DMSO controls.

treatment did not have an additive eﬀect on them (Figure

11D−F). These results further conﬁrm that the eﬀect of DC-2

on the neddylation of cullin3 is through its speciﬁc binding to

DCN1.

Eﬀects of DC-2 on DCN1 Highly Expressed Cancer

Cells. DCN1 is highly expressed in most cancers and has been

discovered as an oncogene.^{10,23,33−39,44−46,69}To determine

whether DC-2 has the ability of inhibiting cancer cells’

proliferation, eight DCN1 ampliﬁed cancer cell lines (KYSE70,

PC9, SMMC-7721, PC3, MCF7, TE-1, KYSE140, and EC109)

and two normal cell lines (GES-1 and L02) were chosen to be

treated with DC-2 for 72 h. As shown in Figure 12A, DC-2

shows strong antiproliferation ability on these cancer cells

rather than normal cells. Furthermore, compound DC-2 could

decrease three types of lung cancer cells’ viabilities (H1975,

PC9, and A549) with time-dependent manner (Figure 12B),

block their colony formations (Figure 12C and D), and induce

their apoptosis dose dependently (Figure 12E and F). These

results suggest that compound DC-2 has the ability of blocking

DCN1 highly expressed cancer cells’ proliferation. In addition,

the cytotoxicities of DC-2N and the compounds with very

similar structures of DC-2N (27, 29, 36, 37, and 45) or DC-2

(33, 39, 40, 46, and 47) on ﬁve DCN1 highly expressed cancer

cells (KYSE70, PC9, SMMC-7721, PC3, and MCF7) were

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Figure 11. Protein levels of DCN1, NRF2, HO-1, NQO1, Cullin1, and Cullin3 were measured after H1975 and PC9 cells were treated with three

DCN1 siRNA (A). Nontargeting siRNA (NT) treatment was used as control. (D) The protein levels of Cullin3, DCN1, NRF2, HO-1, and NQO1

were determined after treating the H1975 and PC9 cells by siRNA#3 with or without compound DC-2. (B, C, E, and F) Densitometry shows

relative protein expression level normalized by GAPDH. Data are presented as means SD. Three individual experiments were performed for each

group. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to the controls (NT).

determined. As shown in Table 6, compounds 33, 39, 40, 46,

and 47, which can block the DCN1−UBE2M interaction at

nanomole level in vitro, also have cytotoxicities on those tested

cancer cells with IC₅₀values ranging from 1 to 12 μM.

However, DC-2N, 27, 29, 36, 37, and 45, which have no

inhibiting eﬀect on DCN1−UBE2M interaction, exhibits less

cytotoxic activity (>20 μM), indicating that the cytotoxicities

of these compounds may be related to their blocking eﬀects on

UBE2M−DCN1 interaction. However, the cytotoxic activities

of DI-591 on the ﬁve DCN1 highly expressed cancer cell lines

were also measured. The IC₅₀values were all above 20 μM at

72 h. On the basis of the current ﬁndings, whether DCN1 can

be recognized as a valuable antitumor target remains to be

further investigated. In addition, except for the inhibitory

eﬀects of DC-2 on DCN1−2, DC-2 also exhibits relatively

weak binding aﬃnities on DCN3−5 (Table 5), similar to the

ﬁndings on NAcM-COV.⁴³Therefore, the cytotoxicities of

DC-2 may be also related to its inhibition on DCN3−5.

However, the relative less cytotoxicity of DI-591 indicates its

higher selectivity on DCN1−2 over DCN3−5.³⁰

CONCLUSIONS

■

In this study, a novel series of compounds with a 5-cyano-6-

phenylpyrimidin scaﬀold as DCN1−UBE2M interaction

modulators have been identiﬁed through structure-based

optimization, which enriches the structure types of DCN1

inhibitors. Among the inhibitors, compound DC-2 exhibits the

most potent inhibition eﬀect on DCN1−UBE2M interaction

at molecule and cellular levels. Molecular docking results show

that DC-2 can well dock into the binding pocket of DCN1.

Site-speciﬁc mutations further veriﬁed its blocking eﬀects.

Furthermore, unlike MLN4924, obliterating all cullins

neddylation, DC-2 speciﬁcally diminishes the neddylation of

cullin3, which leads to the accumulation of cullin3′s substrate

NRF2 and NRF2’s downstream proteins: HO-1 and NQO1.

Our ﬁndings indicate that the 5-cyano-6-phenylpyrimidin-

based small molecules may serve as leading compounds

speciﬁcally targeting DCN1−UBE2M interaction.

EXPERIMENTAL SECTION

■

General Methods for Chemistry. Chemicals and solvents were

obtained from standard suppliers and used directly without further

puriﬁcation. Melting points were taken on an X-5 micromelting

apparatus and were uncorrected. ¹H and ¹³C NMR spectra were

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Figure 12. Compound DC-2 inhibits cell proliferation and colony formation. (A) The inhibition rates (%) of DC-2 on eight diﬀerent cancer cell

lines and two normal cell lines: L02 and GES-1. (B) The IC₅₀values were determined after treating the three lung cancer cell lines (PC9, H1975,

and A549) with DC-2 for 24, 48, and 72 h. (C) After the treatment of compound DC-2 at 0, 0.625, 1.25, 2.5, 5, and 10 μM for 7 days, cells were

stained by crystal violet and then imaged by microscopy. (D) Subsequently, they were dissolved and then measured by a BioTek microplate reader.

(E and F) The apoptosis was determined by ﬂow cytometry after PC9, H1975, and A549 cells were treated with compound DC-2 for 48 h. Data

are presented as means SD. Three individual experiments were performed for each group. *P < 0.05, **P < 0.01, ***P < 0.001 as compared to

the controls.

respectively determined with a 400 and 100 MHz spectrometer. High-

resolution mass spectra (HRMS) were obtained with a Water Q-TOF

electrospray mass spectrometer (Water, Milford, MA). Final products

were of >95% purity as analyzed by HPLC analysis (Phenomenex

column, C-18, 5.0 μm, 4.6 mm × 150 mm) on a Dionex UltiMate

3000 UHPLC instrument from ThermoFisher. Besides, PAINS

screening of the synthesized compounds was carried out by

employing the online program,⁷⁰and all of the tested compounds

passed the ﬁlter.

General Procedure for the Synthesis of Compounds 6−52

and 54. To a well-stirred solution of the appropriate mercapto

heterocyclics and anilines (5 mmol) in absolute ethanol (10 mL), an

equimolar amount of a solution of compounds 5a−o or 53 (5 mmol)

in absolute ethanol (10 mL) was added. The reaction mixture was

stirred and heated under reﬂux for 5 h. Upon completion, the

precipitated product was ﬁltered oﬀ and washed with ethanol to aﬀord

the crude product. The crude product was recrystallized from ethanol

to yield the pure products 6−52 and 54. The detailed information on

the synthesis and characterization of compounds 5a−o and 53 was

reported in published articles.^54,71−73

4-((1-Methyl-1H-tetrazol-5-yl)thio)-6-(2-oxo-2H-chromen-6-yl)-

2-(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (6). Yield 88.5%.

White solid. Mp: 184−185 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 8.36 (d, J = 1.7 Hz, 1H, Ar−H), 8.22 (dd, J = 6.2, 4.0 Hz,

2H, Ar−H), 7.66 (d, J = 14.7 Hz, 1H, Ar−H), 6.62 (d, J = 9.6 Hz, 1H,

Ar−H), 4.14 (s, 3H, −CH₃), 3.57 (d, 2H, −CH₂−), 3.17 (s, 1H, J =

2.5 Hz, C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.31,

169.73, 165.20, 159.34, 155.73, 145.48, 143.73, 132.19, 130.26,

129.64, 119.06, 117.39, 117.10, 114.61, 98.65, 78.79, 73.56, 34.79,

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a

Table 6. Cytotoxic Activities (IC₅₀’s) of DC-2 and DC-2N Derivatives against Five DCN1 Highly Expressed Cancer Cell Lines

b

IC₅₀(μM)

compd

KYSE70

PC9

>20

SMMC-7721

PC3

>20

MCF7

>20

DCN1−UBE2M IC₅₀(nM)-FP

27

29

>20

>10000

>10

>20

>10

>10000

DC-2N

>20

>10000

36

>20

>10000

37

>20

>10000

45

>20

>20000

33

39

40

DC-2

46

47

8.25 0.91

5.35 0.72

10.88 1.03

4.30 0.63

6.56 0.81

5.32 0.72

17.89 1.25

7.96 0.90

4.13 0.61

12.11 1.08

1.11 0.04

5.30 0.72

6.48 0.81

5.59 0.74

4.66 0.66

6.04 0.78

3.99 0.60

2.45 0.39

7.34 0.86

8.64 0.93

16.24 1.21

10.28 1.01

3.62 0.55

8.53 0.93

1.62 0.32

7.17 0.85

3.54 0.55

25.27 1.40

9.82 0.99

3.23 0.51

11.63 1.06

2.20 0.34

6.18 0.79

4.12 0.61

10.17 1.00

195.66 2.29

36.59 0.18

551.87 1.71

26.89 0.24

63.51 1.80

106.21 2.02

ND

NAcM-COV

a

Cells were treated with diﬀerent concentrations of the indicated compounds for 72 h. Cell viability was measured by MTT assay as described in

b

the Experimental Section. IC₅₀values were indicated as the mean

SD of three independent experiments. ND means the value was not

determined.

19.12. HR−MS (ESI) calcd for C₁₉H₁₁N₇NaO₂S₂: [M + Na]⁺m/z,

456.0313; found, 456.0315.

H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.30, 169.69, 165.47,

145.47, 137.36, 132.92, 130.85, 129.06, 114.55, 98.72, 78.78, 73.51,

34.78, 19.10. HR−MS (ESI) calcd for C₁₆H₁₀ClN₇S₂: [M + Na]⁺m/

z, 422.0025; found, 422.0025.

4-((1-Methyl-1H-tetrazol-5-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(thi-

ophen-3-yl)pyrimidine-5-carbonitrile (7). Yield 83.9%. White solid.

1

Mp: 183−184 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ 8.32 (s,

4-(3-Chlorophenyl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-(prop-

1H, Ar−H), 8.13 (d, J = 4.1 Hz, 1H, Ar−H), 7.39 (s, 1H, Ar−H),

4.11 (s, 3H, −CH₃), 3.59 (d, 2H, −CH₂−), 3.15 (t, 1H, C−H).

¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 171.87, 169.80, 158.29,

2-yn-1-ylthio)pyrimidine-5-carbonitrile (12). Yield 89.7%. White

1

solid. Mp: 163−165 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ

8.00 (t, J = 1.8 Hz, 1H, Ar−H), 7.94 (d, J = 7.8 Hz, 1H, Ar−H),

7.78−7.73 (m, 1H, Ar−H), 7.67 (t, J = 7.9 Hz, 1H, Ar−H), 4.13 (s,

3H, −CH₃), 3.60 (d, J = 7.5 Hz, 2H, −CH₂−), 3.16 (t, J = 2.5 Hz,

1H, C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.39,

169.63, 165.23, 145.46, 136.11, 133.55, 131.96, 130.87, 128.65,

127.65, 114.43, 99.11, 78.81, 73.46, 34.77, 19.12. HR−MS (ESI)

calcd for C₁₆H₁₀ClN₇NaS₂: [M + Na]⁺m/z, 422.0025; found,

422.0025.

145.45, 138.13, 135.80, 132.60, 129.72, 114.96, 94.27, 78.77, 73.52,

34.75, 19.06. HR−MS (ESI) calcd for C₁₄H₉N₇NaS₃: [M + Na]⁺m/z,

393.9979; found, 393.9982.

4-(1H-Indol-3-yl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-(prop-2-

yn-1-ylthio)pyrimidine-5-carbonitrile (8). Yield 86.6%. White solid.

Mp: 227−227 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 12.37 (s,

1H, NH, D₂O exchangeable), 8.63 (d, J = 3.0 Hz, 1H, −CH), 8.41

(d, J = 7.8 Hz, 1H, Ar−H), 7.57 (d, J = 7.9 Hz, 1H, Ar−H), 7.29 (t, J

= 7.3 Hz, 1H, Ar−H), 7.24 (t, J = 7.5 Hz, 1H, Ar−H), 4.13 (s, 3H,

−CH₃), 3.59 (d, J = 2.3 Hz, 2H, −CH₂−), 3.17 (t, J = 2.4 Hz, 1H, 

C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 171.68, 168.60,

161.76, 136.52, 132.66, 125.44, 123.43, 122.29, 122.06, 116.14,

112.60, 110.30, 93.65, 79.15, 73.57, 34.75, 18.78. HR−MS (ESI)

calcd for C₁₈H₁₂N₈NaS₂: [M + Na]⁺m/z, 427.0524; found, 427.0525.

4-((1-Methyl-1H-tetrazol-5-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(1H-

pyrrol-2-yl)pyrimidine-5-carbonitrile (9). Yield 51.6%. White solid.

Mp: 189−190 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 12.08 (s,

1H, NH, D₂O exchangeable), 7.49 (s, 1H, −CH), 7.29 (s, 1H,

−CH), 6.53−6.32 (m, 1H, −CH), 4.10 (s, 3H, −CH₃), 3.69 (d,

J = 2.6 Hz, 2H, −CH₂−), 3.15 (t, J = 2.5 Hz, 1H, C−H). ¹³C NMR

(100 MHz, DMSO-d₆, ppm): δ 171.53, 168.77, 155.43, 145.62,

127.32, 125.45, 117.04, 115.44, 112.13, 91.76, 79.03, 73.67, 34.72,

18.86. HR−MS (ESI) calcd for C₁₄H₁₀N₈NaS₂: [M + Na]⁺m/z,

418.0521; found, 418.0521.

4-((1-Methyl-1H-tetrazol-5-yl)thio)-2-(prop-2-yn-1-ylthio)-6-

(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (10). Yield

82.1%. White solid. Mp: 256−256 °C. ¹H NMR (400 MHz,

DMSO-d₆, ppm): δ 7.40 (s, 2H, Ar−H), 4.12 (s, 3H, −CH₃), 3.87

(s, 6H, −CH₃), 3.80 (s, 3H, −CH₃), 3.65 (t, J = 12.7 Hz, 2H,

−CH₂−), 3.20 (t, J = 2.3 Hz, 1H, C−H). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 172.55, 170.08, 166.35, 153.39, 146.06, 141.54,

129.54, 115.48, 107.34, 98.83, 79.69, 73.84, 60.78, 56.69, 35.24, 19.77.

HR−MS (ESI) calcd for C₁₉H₁₇N₇NaO₃S₂: [M + Na]⁺m/z,

478.0732; found, 478.0733.

4-(4-Bromophenyl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-(prop-

2-yn-1-ylthio)pyrimidine-5-carbonitrile (13). Yield 78.1%. Yellow

1

solid. Mp: 238−239 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ

8.23 (d, J = 3.3 Hz, 1H, −CH), 8.13 (d, J = 3.3 Hz, 1H, −CH),

7.97−7.91 (m, 2H, Ar−H), 7.88−7.82 (m, 2H, Ar−H), 3.79 (d, J =

2.6 Hz, 2H, −CH₂−), 3.14 (t, J = 2.6 Hz, 1H, C−H). ¹³C NMR

(100 MHz, DMSO-d₆, ppm): δ 172.03, 171.48, 165.43, 150.29,

144.31, 133.50, 131.91, 131.04, 127.91, 126.18, 114.45, 98.20, 79.06,

73.86, 19.29. HR−MS (ESI) calcd for C₁₆H₁₀BrN₇NaS₂: [M + Na]⁺

m/z, 466.9070; found, 466.9073.

4-((1-Methyl-1H-tetrazol-5-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(p-

tolyl)pyrimidine-5-carbonitrile (14). Yield 83.1%. Yellow solid. Mp:

1

164−165 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ 7.92 (d, J =

8.2 Hz, 2H, Ar−H), 7.44 (d, J = 8.1 Hz, 2H, Ar−H), 4.13 (s, 3H,

−CH₃), 3.56 (t, J = 20.3 Hz, 2H, −CH₂−), 3.15 (t, J = 2.5 Hz, 1H, 

C−H), 2.42 (s, 3H, −CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ppm):

δ 172.13, 169.62, 166.39, 145.54, 142.88, 131.32, 129.49, 129.01,

114.82, 98.19, 78.87, 73.46, 34.76, 21.09, 19.05. HR−MS (ESI) calcd

for C₁₇H₁₃N₇NaS₂: [M + Na]⁺m/z, 402.0572; found, 402.0573.

4-(4-Isopropylphenyl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-

(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (15). Yield 83.4%.

White solid. Mp: 141−142 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.95 (d, J = 3.9 Hz, 2H, Ar−H), 7.50 (d, J = 8.0 Hz, 2H, Ar−

H), 4.14 (d, J = 4.0 Hz, 3H, −CH₃), 3.60 (s, 2H, −CH₂−), 3.15 (t, J

= 2.5 Hz, 1H, C−H), 3.05−2.96 (m, 1H, −CH), 1.25 (d, J = 6.8

Hz, 6H, −CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.16,

169.65, 166.36, 153.39, 145.54, 131.70, 129.19, 126.92, 114.85, 98.17,

78.89, 73.46, 34.77, 33.47, 23.47, 19.06. HR−MS (ESI) calcd for

C₁₉H₁₇N₇NaS₂: [M + Na]⁺m/z, 430.0885; found, 430.0886.

4-(4-Chlorophenyl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-(prop-

2-yn-1-ylthio)pyrimidine-5-carbonitrile (11). Yield 87.7%. White

1

solid. Mp: 163−164 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ

4-(3-Methoxyphenyl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-

(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (16). Yield 89.9%.

White solid. Mp: 189−192 °C. ¹H NMR (400 MHz, DMSO-d₆,

8.02 (d, J = 8.4 Hz, 2H, Ar−H), 7.74 (t, J = 19.1 Hz, 2H, Ar−H), 4.13

(s, 3H, −CH₃), 3.59 (s, 2H, −CH₂−), 3.15 (s, J = 2.5 Hz, 1H, C−

N

DOI: 10.1021/acs.jmedchem.9b00003

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Journal of Medicinal Chemistry

Article

ppm): δ 7.56 (s, 1H, Ar−H), 7.54 (d, J = 3.7 Hz, 2H, Ar−H), 7.25

(dt, J = 6.9, 2.2 Hz, 1H, Ar−H), 4.12 (s, 3H, −CH₃), 3.84 (s, 3H,

−CH₃), 3.61 (d, J = 2.4 Hz, 2H, −CH₂−), 3.17 (t, J = 2.5 Hz, 1H, 

C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.22, 169.63,

166.35, 159.27, 145.52, 135.41, 130.15, 121.20, 118.09, 114.67,

114.17, 98.78, 78.95, 73.42, 55.43, 34.76, 19.11. HR−MS (ESI) calcd

for C₁₇H₁₃N₇ONaS₂: [M + Na]⁺m/z, 418.0521; found, 418.0521.

4-(3,4-Dichlorophenyl)-6-((1-methyl-1H-tetrazol-5-yl)thio)-2-

(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (17). Yield 89.8%.

White solid. Mp: 151−152 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 8.21 (d, J = 1.8 Hz, 1H, Ar−H), 7.97 (dd, J = 8.4, 1.9 Hz,

1H, Ar−H), 7.93 (d, J = 8.4 Hz, 1H, Ar−H), 4.13 (s, 3H, −CH₃),

3.61 (d, J = 2.3 Hz, 2H, −CH₂−), 3.16 (t, J = 2.4 Hz, 1H, C−H).

¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.45, 169.65, 164.28,

(m, 4H, −CH₂−), 3.86 (s, 6H, −CH₃), 3.77 (s, 3H, −CH₃), 3.17 (t, J

= 2.5 Hz, 1H, C−H), 1.68 (s, 6H, −CH₂−). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 170.55, 169.37, 161.46, 152.54, 140.16, 131.04,

118.01, 107.19, 83.42, 80.48, 72.81, 60.15, 56.16, 47.99, 25.58, 23.64,

19.16. HR−MS (ESI) calcd for C₂₂H₂₅N₄O₃S: [M + H]⁺m/z,

425.1647; found, 425.1648.

4-((1-(2-(Dimethylamino)ethyl)-1H-tetrazol-5-yl)thio)-2-(prop-2-

yn-1-ylthio)-6-(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile

1

(26). Yield 75.9%. Yellow solid. Mp: 197−198 °C. H NMR (400

MHz, DMSO-d₆, ppm): δ 7.42 (s, 2H, Ar−H), 4.99 (t, J = 6.6 Hz,

2H, −CH₂−), 3.87 (s, 6H, −CH₃), 3.79 (s, 3H, −CH₃), 3.72 (d, J =

2.3 Hz, 2H, −CH₂−), 3.69 (t, J = 6.6 Hz, 2H, −CH₂−), 3.25 (t, J =

2.5 Hz, 1H, C−H), 2.83 (s, 6H, −CH₃). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 171.96, 169.87, 165.62, 152.90, 146.30, 141.06,

129.01, 115.03, 106.80, 98.30, 79.26, 73.45, 60.29, 56.19, 54.14, 42.69,

42.38, 19.36. HR−MS (ESI) calcd for C₂₂H₂₅N₈O₃S₂: [M + H]⁺m/z,

513.1491; found, 513.1491.

145.42, 135.21, 134.56, 131.82, 131.26, 130.80, 129.05, 114.32, 99.14,

78.77, 73.52, 34.78, 19.18. HR−MS (ESI) calcd for C₁₆H₉Cl₂N₇NaS₂:

[M + Na]⁺m/z, 455.9636; found, 455.9637.

4-((1H-1,2,4-Triazol-3-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (27). Yield 80.9%.

White solid. Mp: 234−235 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 8.85 (s, 1H, NH, D₂O exchangeable), 7.41 (s, 2H, Ar−H),

3.87 (s, 6H, −CH₃), 3.79 (s, 3H, −CH₃), 3.78−3.78 (s, 2H,

−CH₂−), 3.15 (t, J = 2.4 Hz, 1H, C−H). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 172.22, 169.63, 166.35, 159.27, 145.52, 135.41,

130.15, 121.20, 118.09, 114.67, 114.17, 98.78, 78.95, 73.42, 55.43,

34.76, 19.11. HR−MS (ESI) calcd for C₁₉H₁₆N₆NaO₃S₂: [M + Na]⁺

m/z, 463.0623; found, 463.0622.

4-((1-Methyl-1H-imidazol-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-

(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (28). Yield

32.8%. White solid. Mp: 176−177 °C. ¹H NMR (400 MHz,

DMSO-d₆, ppm): δ 7.62 (s, 1H, −CH), 7.38 (s, 2H, Ar−H),

7.22 (s, 1H, −CH), 3.87 (s, 6H, −CH₃), 3.79 (s, 3H, −CH₃), 3.68

(s, 2H, −CH₂−), 3.67 (s, 3H, −CH₃), 3.20 (t, J = 2.2 Hz, 1H, C−

H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 173.22, 171.82, 165.81,

152.83, 140.84, 130.39, 130.16, 129.27, 126.44, 115.07, 106.86, 97.48,

79.44, 73.36, 60.25, 56.18, 33.68, 19.22. HR−MS (ESI) calcd for

C₂₁H₁₉N₅NaO₃S₂: [M + Na]⁺m/z, 476.0827; found, 476.0828.

2-(Prop-2-yn-1-ylthio)-4-(pyridin-2-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (29). Yield 80.2%.

Yellow solid. Mp: 180−181 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 8.76−8.58 (m, 1H, Ar−H), 7.97 (td, J = 7.7, 1.7 Hz, 1H, Ar−

H), 7.86 (d, J = 7.8 Hz, 1H, Ar−H), 7.55 (dt, J = 15.7, 7.8 Hz, 1H,

Ar−H), 7.38 (s, 2H, Ar−H), 3.86 (s, 6H, −CH₃), 3.78 (s, 3H,

−CH₃), 3.71 (d, J = 2.2 Hz, 2H, −CH₂−), 3.14 (t, J = 2.3 Hz, 1H).

¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.90, 171.53, 165.73,

4-((1-Methyl-1H-tetrazol-5-yl)thio)-6-phenyl-2-(prop-2-yn-1-

ylthio)pyrimidine-5-carbonitrile (18). Yield 81.9%. White solid. Mp:

1

130−131 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ 8.17 (d, J =

7.5 Hz, 2H, Ar−H), 8.07 (s, 1H, Ar−H), 7.59−7.55 (m, 2H, Ar−H),

4.10 (s, 3H, −CH₃), 3.67 (d, 2H, −CH₂−), 3.11 (t, J = 2.5 Hz,1H, 

C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.30, 169.69,

165.47, 145.47, 137.36, 132.92, 130.85, 129.06, 114.55, 98.72, 78.78,

73.51, 34.78, 19.10. HR−MS (ESI) calcd for C₁₆H₁₁N₇NaS₂: [M +

Na]⁺m/z, 388.0415; found, 388.0415.

4-((1-Methyl-1H-tetrazol-5-yl)amino)-2-(prop-2-yn-1-ylthio)-6-

(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (19). Yield

94.1%. Yellow solid. Mp: 205−206 °C. ¹H NMR (400 MHz,

DMSO-d₆, ppm): δ 7.38 (s, 2H, Ar−H), 3.93 (s, 3H, −CH₃), 3.87 (s,

6H, −CH₃), 3.85 (d, J = 1.8 Hz, 2H, −CH₂−), 3.78 (s, 3H, −CH₃),

3.16 (t, J = 2.5 Hz, 1H, C−H). ¹³C NMR (100 MHz, DMSO-d₆,

ppm): δ 171.96, 167.33, 161.41, 152.71, 150.04, 140.43, 130.11,

115.42, 106.68, 86.40, 79.95, 72.99, 60.22, 56.11, 33.61, 19.17. HR−

MS (ESI) calcd for C₁₉H₁₈N₈NaO₃S: [M + Na]⁺m/z, 461.1120;

found, 461.1122.

4-(Piperazin-1-yl)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (20). Yield 89.0%.

White solid. Mp: 242−243 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 9.72 (s, 1H, NH, D₂O exchangeable), 7.30 (s, 2H, Ar−H),

4.19 (s, 4H, −CH₂−), 4.03 (d, J = 2.1 Hz, 2H, −CH₂−), 3.86 (s, 6H,

−CH₃), 3.77 (s, 3H, −CH₃), 3.34 (s, 4H, −CH₂−), 3.21 (t, J = 2.3

Hz, 1H, C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 170.58,

169.15, 161.75, 152.60, 140.23, 130.88, 117.90, 107.18, 83.78, 80.45,

73.00, 60.18, 56.16, 45.75, 19.26. HR−MS (ESI) calcd for

C₂₁H₂₄N₅O₃S: [M + H]⁺m/z, 426.1600; found, 426.1602.

2-(Prop-2-yn-1-ylthio)-4-thiomorpholino-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (21). Yield 50.9%.

Yellow solid. Mp: 152−153 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.28 (s, 2H, Ar−H), 4.19 (m, 4H, −CH₂−), 3.98 (d, J = 2.4

Hz, 2H, −CH₂−), 3.85 (s, 6H, −CH₃), 3.77 (s, 3H, −CH₃), 3.19 (t, J

= 2.3 Hz, 1H, C−H), 2.86−2.77 (m, 4H, −CH₂−). ¹³C NMR (100

MHz, DMSO-d₆, ppm): δ 170.71, 169.42, 161.97, 152.56, 140.21,

130.93, 117.92, 107.23, 84.24, 80.46, 72.89, 60.17, 56.17, 49.70, 26.50,

19.22. HR−MS (ESI) calcd for C₂₁H₂₂N₄O₃NaS₂: [M + Na]⁺m/z,

465.1031; found, 465.1032.

152.80, 150.76, 149.32, 140.76, 138.08, 131.26, 129.36, 124.71,

115.22, 106.82, 98.15, 79.54, 73.35, 60.24, 56.14, 19.20. HR−MS

(ESI) calcd for C₂₂H₁₈N₄NaO₃S₂: [M + Na]⁺m/z, 473.0718; found,

473.0715.

4-((5-Aminothiophen-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (30). Yield 89.3%.

Yellow solid. Mp: 189−190 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.87 (s, 2H, −CH), 7.42 (s, 2H, Ar−H), 3.95 (d, J = 2.1

Hz, 2H, −CH₂−), 3.86 (s, 6H, −CH₃), 3.79 (s, 3H, −CH₃), 3.17 (d, J

= 2.2 Hz, 1H, C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ

173.78, 171.83, 171.36, 165.58, 152.84, 140.98, 129.19, 114.91,

106.95, 97.68, 79.43, 73.44, 60.27, 56.19, 19.55. HR−MS (ESI) calcd

for C₂₁H₁₈N₄NaO₃S₃: [M + Na]⁺m/z, 493.0439; found, 493.0438.

4-((5-Methyl-1,3,4-thiadiazol-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-

(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (31). Yield

76.5%. White solid. Mp: 186−187 °C. ¹H NMR (400 MHz,

DMSO-d₆, ppm): δ 7.44 (s, 2H, Ar−H), 3.93 (d, J = 2.4 Hz, 2H,

−CH₂−), 3.87 (s, 6H, −CH₃), 3.80 (s, 3H, −CH₃), 3.20 (t, J = 2.5

Hz, 1H, C−H), 2.86 (s, 1H, −CH₃). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 171.88, 171.26, 169.59, 165.64, 154.06, 152.86,

141.07, 129.09, 114.85, 106.96, 98.14, 79.53, 73.43, 60.27, 56.19,

19.54, 15.53. HR−MS (ESI) calcd for C₂₀H₁₇N₅NaO₃S₃: [M + Na]⁺

m/z, 494.0391; found, 494.0389.

4 - M o r p h o l i n o - 2 - ( p r o p - 2 - y n - 1 - y l t h i o ) - 6 - ( 3 , 4 , 5 -

trimethoxyphenyl)pyrimidine-5-carbonitrile (22). Yield 89.5%.

White solid. Mp: 163−164 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.27 (s, 2H, Ar−H), 3.98 (d, J = 2.5 Hz, 2H, −CH₂−),

3.97−3.93 (m, 4H, −CH₂−), 3.85 (s, 6H, −CH₃), 3.76 (s, 3H,

−CH₃), 3.74 (m, 4H, −CH₂−), 3.19 (t, J = 2.5 Hz, 1H, C−H). ¹³

C

NMR (100 MHz, DMSO-d₆, ppm): δ 170.70, 169.34, 161.84, 152.58,

140.24, 130.87, 117.86, 107.20, 83.94, 80.40, 72.91, 65.91, 60.17,

56.16, 47.17, 19.23. HR−MS (ESI) calcd for C₂₁H₂₃N₄O₄S: [M +

H]⁺m/z, 427.1440; found, 427.1441.

4-(Piperidin-1-yl)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (23). Yield 90.0%.

White solid. Mp: 120−121 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.27 (s, 2H, Ar−H), 3.97 (d, J = 2.5 Hz, 2H, −CH₂−), 3.90

4-((5-Amino-1,3,4-thiadiazol-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-

(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (32). Yield

65.1%. White solid. Mp: 181−182 °C. ¹H NMR (400 MHz,

O

DOI: 10.1021/acs.jmedchem.9b00003

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Journal of Medicinal Chemistry

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DMSO-d₆, ppm): δ 7.83 (s, 2H, NH, D₂O exchangeable), 7.43 (d, J =

0.6 Hz, 2H, Ar−H), 3.94 (t, J = 7.4 Hz, 2H, −CH₂−), 3.87 (s, 6H,

−CH₃), 3.79 (d, J = 0.8 Hz, 3H, −CH₃), 3.18 (dd, J = 2.5, 1.6 Hz,

1H, C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 173.82,

171.81, 171.41, 165.59, 152.84, 140.96, 138.79, 129.21, 114.92,

106.95, 97.71, 79.44, 73.48, 60.26, 56.19, 19.55.

4-((1,3,4-Thiadiazol-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (33). Yield 81.1%.

White solid. Mp: 228−229 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 9.96 (s, 1H, −CH), 7.45 (s, 2H, Ar−H), 3.94 (d, J = 2.5

Hz, 2H, −CH₂−), 3.87 (s, 6H, −CH₃), 3.79 (s, 3H, −CH₃), 3.20 (t, J

= 2.5 Hz, 1H, C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ

171.83, 169.14, 165.64, 159.31, 155.09, 152.88, 141.10, 129.10,

114.85, 106.99, 98.24, 79.51, 73.53, 60.28, 56.20, 19.57.

−CH₂−), 3.15 (t, J = 2.5 Hz, 1H, C−H). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 172.00, 171.47, 165.31, 150.28, 144.31, 137.20,

133.13, 130.92, 128.97, 127.92, 114.47, 98.23, 79.08, 73.86, 19.27.

HR-MS (ESI) calcd for C₁₇H₉ClN₄NaS₃: [M + Na]⁺m/z, 422.9576;

found, 422.9578.

4-(4-Bromophenyl)-2-(prop-2-yn-1-ylthio)-6-(thiazol-2-ylthio)-

pyrimidine-5-carbonitrile (40). Yield 78.1%. White solid. Mp: 238−

239 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.23 (d, J = 3.3 Hz,

1H, −CH), 8.13 (d, J = 3.3 Hz, 1H, −CH), 7.97−7.91 (m, 2H,

Ar−H), 7.88−7.82 (m, 2H, Ar−H), 3.79 (d, J = 2.6 Hz, 2H,

−CH₂−), 3.14 (t, J = 2.6 Hz, 1H, C−H). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 172.03, 171.48, 165.43, 150.29, 144.31, 133.50,

131.91, 131.04, 127.91, 126.18, 114.45, 98.20, 79.06, 73.86, 19.29.

HR−MS (ESI) calcd for C₁₇H₉BrN₄NaS₃: [M + Na]⁺m/z, 466.9070;

found, 466.9073.

2-(Prop-2-yn-1-ylthio)-4-(thiazol-2-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (34). Yield 81.1%.

White solid. Mp: 172−173 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): 8.20 (d, J = 3.2 Hz, 1H, −CH), 8.11 (d, J = 3.2 Hz, 1H,

−CH), 7.42 (s, 2H, Ar−H), 3.87 (s, 6H, −CH₃), 3.85 (s, 2H,

−CH₂−), 3.79 (s, 3H, −CH₃), 3.18 (s, 1H, C−H). ¹³C NMR (100

MHz, DMSO-d₆, ppm): δ 171.75, 171.21, 165.61, 152.84, 150.61,

144.13, 140.94, 129.20, 127.64, 114.88, 106.93, 97.72, 79.49, 79.11,

73.49, 60.26, 56.17, 19.41. HR−MS (ESI) calcd for

C₂₀H₁₆N₄NaO₃S₃: [M + Na]⁺m/z, 479.0282; found, 479.0284.

4-((4-Methylthiazol-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (35). Yield 76.7%.

White solid. Mp: 226−227 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.75 (d, J = 0.9 Hz, 1H, −CH), 7.41 (s, 2H, Ar−H), 3.86

(s, 6H, −CH₃), 3.86 (d, J = 2.8 Hz, 2H, −CH₂−), 3.79 (s, 3H,

4-(3,4-Dichlorophenyl)-2-(prop-2-yn-1-ylthio)-6-(thiazol-2-

ylthio)pyrimidine-5-carbonitrile (41). Yield 88.0%. White solid. Mp:

1

142−143 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ 8.23 (d, J =

3.1 Hz, 2H, Ar−H), 8.13 (d, J = 3.3 Hz, 1H, Ar−H), 7.98 (dd, J = 8.4,

1.7 Hz, 1H, −CH), 7.91 (d, J = 8.4 Hz, 1H, −CH), 3.80 (d, J =

2.3 Hz, 2H, −CH₂−), 3.15 (s, 1H, C−H). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 172.14, 171.46, 164.05, 150.12, 144.36, 135.06,

134.74, 131.74, 131.14, 130.89, 129.11, 127.99, 114.26, 98.58, 79.06,

73.84, 19.35. HR−MS (ESI) calcd for C₁₇H₈C_l2N₄NaS₃: [M + Na]⁺

m/z, 456.9186; found, 456.9187.

2-(Prop-2-yn-1-ylthio)-4-(thiazol-2-ylthio)-6-(p-tolyl)pyrimidine-

1

5-carbonitrile (42). Yield 72.8%. White solid. Mp: 156−157 °C. H

NMR (400 MHz, DMSO-d₆, ppm): δ 8.21 (d, J = 3.3 Hz, 1H,

−CH), 8.12 (d, J = 3.3 Hz, 1H, −CH), 7.93 (d, J = 8.1 Hz, 2H,

Ar−H), 7.43 (d, J = 8.1 Hz, 2H, Ar−H), 3.80 (d, J = 2.5 Hz, 2H,

−CH₂−), 3.15 (t, J = 2.4 Hz, 1H, C−H), 2.41 (d, J = 6.0 Hz, 3H,

−CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.22, 169.63,

166.35, 159.27, 145.52, 135.41, 130.15, 121.20, 118.09, 114.67,

114.17, 98.78, 78.95, 73.42, 55.43, 34.76, 19.11. HR−MS (ESI) calcd

for C₁₈H₁₂N₄NaS₃: [M + Na]⁺m/z, 403.0122; found, 403.0123.

4-(2-Oxo-2H-chromen-6-yl)-2-(prop-2-yn-1-ylthio)-6-(thiazol-2-

ylthio)pyrimidine-5-carbonitrile (43). Yield 90.8%. Yellow solid. Mp:

−CH₃), 3.18 (t, J = 2.6 Hz, 1H), 2.46 (d, J = 0.8 Hz, 3H, −CH₃). ¹³

C

NMR (100 MHz, DMSO-d₆, ppm): δ 171.72, 171.36, 165.67, 153.78,

152.84, 149.35, 140.94, 129.23, 121.97, 114.88, 106.95, 97.72, 79.51,

73.41, 60.26, 56.19, 19.38, 16.73. HR−MS (ESI) calcd for

C₂₁H₁₈N₄NaO₃S₃: [M + Na]⁺m/z, 493.0439; found, 493.0434.

4-((4,5-Dimethylthiazol-2-yl)thio)-2-(prop-2-yn-1-ylthio)-6-

(3,4,5-trimethoxyphenyl)pyrimidine-5-carbonitrile (36). Yield

82.9%. White solid. Mp: 192−193 °C. ¹H NMR (400 MHz,

DMSO-d₆, ppm): δ 7.23 (s, 2H, Ar−H), 3.70 (s, 6H, −CH₃), 3.62

(s, 3H, −CH₃), 3.22 (s, 2H, −CH₂−), 3.02 (s, 1H, C−H), 2.29 (s,

3H, −CH₃), 2.19 (s, 3H, −CH₃). ¹³C NMR (100 MHz, DMSO-d₆,

ppm): δ 171.72, 171.36, 165.67, 153.78, 152.84, 149.35, 140.94,

129.23, 121.97, 114.88, 106.95, 97.72, 79.51, 73.41, 60.26, 56.19,

19.38, 16.73, 19.24. HR−MS (ESI) calcd for C₂₂H₂₁N₄O₃S₃: [M +

H]+ m/z, 485.0776; found, 485.0774.

1

226−227 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ 8.37 (d, J =

1.9 Hz, 1H, Ar−H), 8.23 (d, J = 3.3 Hz, 1H, Ar−H), 8.22 (d, J = 2.5

Hz, 1H, −CH), 8.20 (s, 1H, −CH), 8.13 (d, J = 3.3 Hz, 1H,

−CH), 7.64 (d, J = 8.7 Hz, 1H, −CH), 6.62 (d, J = 9.6 Hz, 1H,

Ar−H), 3.80 (d, J = 2.4 Hz, 2H, −CH₂−), 3.16 (t, J = 2.4 Hz, 1H, 

C−H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.02, 171.52,

165.02, 159.35, 155.67, 150.23, 144.34, 143.75, 132.30, 130.47,

129.70, 127.97, 118.99, 117.34, 117.01, 114.53, 98.15, 79.08, 73.94,

19.32. HR−MS (ESI) calcd for C₂₀H₁₀N₄O₂NaS₃: [M + Na]⁺m/z,

456.9864; found, 456.9862.

4-(Benzo[d]thiazol-2-ylthio)-2-(prop-2-yn-1-ylthio)-6-(3,4,5-

trimethoxyphenyl)pyrimidine-5-carbonitrile (37). Yield 69.0%.

White solid. Mp: 223−224 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 8.25−8.17 (m, 1H, Ar−H), 8.10 (d, J = 7.6 Hz, 1H, Ar−H),

7.58 (dtd, J = 16.4, 7.3, 1.3 Hz, 2H, Ar−H), 7.47 (s, 2H, Ar−H), 3.92

(d, J = 2.4 Hz, 2H, −CH₂−), 3.88 (s, 6H, −CH₃), 3.80 (s, 3H,

−CH₃), 3.15 (t, J = 2.5 Hz, 1H, C−H). ¹³C NMR (100 MHz,

DMSO-d₆, ppm): δ 171.72, 169.72, 165.59, 155.24, 152.86, 151.49,

141.09, 136.73, 129.15, 126.81, 126.23, 122.92, 122.16, 114.86,

107.01, 98.14, 79.40, 73.39, 60.27, 56.20, 19.52. HR−MS (ESI) calcd

for C₂₄H₁₈N₄NaO₃S₃: [M + Na]⁺m/z, 529.0439; found, 529.0439.

4-(3-Chlorophenyl)-2-(prop-2-yn-1-ylthio)-6-(thiazol-2-ylthio)-

pyrimidine-5-carbonitrile (38). Yield 65.4%. White solid. Mp: 171−

172 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.24−8.21 (m, 1H,

Ar−H), 8.13 (d, J = 2.9 Hz, 1H, Ar−H), 8.03 (s, 1H, Ar−H), 7.96 (d,

J = 7.6 Hz, 1H, Ar−H), 7.75 (d, J = 8.0 Hz, 1H, −CH), 7.66 (t, J =

7.8 Hz, 1H, −CH), 3.80 (s, 2H, −CH₂−), 3.15 (s, 1H, C−H).

¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.10, 171.43, 165.05,

150.21, 144.34, 136.33, 133.49, 131.82, 130.75, 128.75, 127.96,

127.72, 114.35, 98.60, 79.11, 73.82, 19.31. HR-MS (ESI) calcd for

C₁₇H₉ClN₄NaS₃: [M + Na]⁺m/z, 422.9576; found, 422.9577.

4-(4-Chlorophenyl)-2-(prop-2-yn-1-ylthio)-6-(thiazol-2-ylthio)-

pyrimidine-5-carbonitrile (39). Yield 73.2%. White solid. Mp: 136−

137 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 8.21 (t, J = 10.7 Hz,

1H, −CH), 8.13 (d, J = 3.3 Hz, 1H, −CH), 8.03 (d, J = 8.6 Hz,

2H, Ar−H), 7.71 (d, J = 8.6 Hz, 2H, Ar−H), 3.79 (d, J = 2.4 Hz, 2H,

2-(Prop-2-yn-1-ylthio)-4-(pyridin-4-yl)-6-(thiazol-2-ylthio)-

pyrimidine-5-carbonitrile (44). Yield 76.3%. White solid. Mp: 189−

1

190 °C. H NMR (400 MHz, DMSO-d₆, ppm): δ 8.87 (dd, J = 4.5,

1.5 Hz, 2H, Ar−H), 8.24 (d, J = 3.3 Hz, 1H, −CH), 8.14 (d, J = 3.3

Hz, 1H, −CH), 7.91 (dd, J = 4.5, 1.6 Hz, 2H, Ar−H), 3.79 (d, J =

2.5 Hz, 2H, −CH₂−), 3.14 (t, J = 2.6 Hz, 1H, C−H). ¹³C NMR

(100 MHz, DMSO-d₆, ppm): δ 190.70, 176.34, 166.45, 165.53,

149.87, 142.64, 140.34, 121.31, 118.64, 117.01, 96.91, 76.90, 73.97,

25.02. HR−MS (ESI) calcd for C₁₆H₉N₅NaS₃: [M + Na]⁺m/z,

389.9918; found, 389.9919.

2-(Prop-2-yn-1-ylthio)-4-(1H-pyrrol-2-yl)-6-(thiazol-2-ylthio)-

pyrimidine-5-carbonitrile (45). Yield 80.1%. White solid. Mp: 255−

256 °C. ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 12.05 (s, 1H, NH,

D₂O exchangeable), 8.17 (d, J = 3.3 Hz, 1H, −CH), 8.08 (d, J = 3.3

Hz, 1H, −CH), 7.56−7.43 (m, 1H, −CH), 7.30 (s, 1H,

−CH), 6.42 (dt, J = 4.2, 2.3 Hz, 1H, −CH), 3.94 (d, J = 2.5 Hz,

2H, −CH₂−), 3.15 (t, J = 2.6 Hz, 1H, C−H). ¹³C NMR (100

MHz, DMSO-d₆, ppm): δ 171.25, 170.38, 155.31, 150.94, 143.92,

127.38, 127.03, 125.56, 116.89, 115.36, 112.01, 91.23, 79.30, 73.98,

19.04. HR−MS (ESI) calcd for C₁₅H₉N₅NaS₃: [M + Na]⁺m/z,

377.9918; found, 377.9917.

4-(4-Chlorophenyl)-6-((1-(2-(dimethylamino)ethyl)-1H-tetrazol-

5-yl)thio)-2-(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (46).

P

DOI: 10.1021/acs.jmedchem.9b00003

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Article

1

Yield 89.0%. White solid. Mp: 194−196 °C. H NMR (400 MHz,

DMSO-d₆, ppm): δ 8.00 (d, J = 8.5 Hz, 2H, Ar−H), 7.69 (d, J = 8.5

Hz, 2H, Ar−H), 4.94 (t, J = 6.1 Hz, 2H, −CH₂−), 3.61 (s, 4H,

−CH₂−), 3.10 (s, 1H, C−H), 2.85 (s, 6H, −CH₃). ¹³C NMR (100

MHz, DMSO-d₆, ppm): δ 172.32, 169.75, 165.42, 146.12, 137.45,

132.85, 130.83, 129.10, 114.52, 98.73, 78.84, 73.55, 54.27, 42.58,

19.16. HR−MS (ESI) calcd for C₁₉H₁₈ClN₈S₂: [M + H]⁺m/z,

457.0784; found, 457.0785.

32.44, 21.92, 13.05. HR−MS (ESI) calcd for C₁₇H₁₃ClN₄NaS₃: [M +

Na]⁺m/z, 426.9889; found, 426.9890.

2-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)thio)-4-(4-chlorophen-

yl)-6-(thiazol-2-ylthio)pyrimidine-5-carbonitrile (54). Yield 32.6%.

White solid. Mp: 177−178 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 8.08 (d, J = 3.3 Hz, 1H, −CH), 7.99 (d, J = 2.4 Hz, 2H,

Ar−H), 7.97 (s, 1H, Ar−H), 7.87 (s, 1H, Ar−H), 7.69 (d, J = 8.6 Hz,

2H, Ar−H), 7.36 (td, J = 8.8, 4.4 Hz, 3H, Ar−H), 7.30−7.23 (m, 2H,

Ar−H), 5.53 (s, 2H, S−CH₂−), 4.24 (s, 2H, −CH₂−). ¹³C NMR

(100 MHz, DMSO-d₆, ppm): δ 190.71, 176.32, 166.40, 165.5, 142.63,

134.33, 133.78, 130.78, 129.37, 128.96, 128.63, 127.62, 125.79,

122.96, 118.62, 117.05, 96.95, 57.32, 35.20. HR-MS (ESI) calcd for

C₂₄H₁₆ClN₇NaS₃: [M + Na]⁺m/z, 556.0216; found, 556.0217.

Reagents. Rabbit monoclonal antibodies against DCN1

(ab181233), Cullin1 (ab75817), Cullin2 (ab166917), Cullin3

(ab75851), UBE2M (ab109507), p21 (ab109520), and Cullin5

(ab184177) were purchased from Abcam Biotechnology (Cambridge,

UK). Anti-DCN1 (GWB-E3D700) antibody was from Genway

Biotech (San Diego, CA). Mouse monoclonal antibody against

CyclinE1 (4129) was purchased from Cell Signaling Technologies.

Rabbit polyclonal antibodies against CDT1 (14382-1-AP), HO-1/

HMOX1 (10701-1-AP), NQO1 (11451-1-AP), NRF2 (16396-1-AP),

CUL4A (14851-1-AP), and CUL4B (12916-1-AP) were purchased

from Proteintech (Wuhan, China).

E. coli DH5α and BL21 (DE3) were obtained from Life

technologies. E. coli BL21-AI was purchased from Invitrogen.

Kanamycin, isopropyl-β-^D-galactopyranoside (IPTG) (I8070), L-

(+)-arabinose (L8060), bicinchoninic acid (BCA) Protein Assay kit

(PC0020), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) (M8180), and glutathione were purchased from

Solarbio (China). Ni-beads and GST-trap column were purchased

from QIAGEN (U.S.). Annexin-V-FITC/PI apoptosis kit (KGA106)

was purchased from Keygen Biotech (China). Enhanced chemilumi-

nescence (QL228436) and Lipofectamine RNAiMAX Reagent

(13778030) were purchased from Thermo Fisher Scientiﬁc. HTRF

detection buﬀer (62SDBRDD), streptavidin-XL665 (610SAXLA),

and anti-GST-cryptate gold (61GSTKLA) were purchased from

Cisbio Bioassays. NEDD8 Conjugation Initiation Kit was obtained

from Boston BioChem (Cambridge, MA). 5000×Sypro Orange dye

was purchased from Invitrogen. Super Streptavidin (SSA) sensor was

purchased from Fortebio.

Cell Lines. Cells were cultivated in corresponding medium

supplemented with 10% FBS and 5% CO₂at 37 °C. Human

esophageal carcinoma cell lines EC109, EC9706, TE-1, human lung

carcinoma cell lines PC9, A549, and H1975, prostate carcinoma cell

line PC3, and liver carcinoma cell lines BEL-7402, SMMC-7721, and

ZIP177 were obtained from Cell Bank of the Chinese Academy of

Sciences (Shanghai, China). Human esophageal carcinoma cell lines

KYSE-70 and KYSE-140 and human immortalized normal esophageal

epithelial cell Het-1A were obtained as gifts from the First Aﬃliated

Hospital of Zhengzhou University, which were purchased from the

American Type Culture Collection (Manassas, VA). Human gastric

epithelial mucosa cell line GES-1 and human breast cancer cell line

MCF-7 were purchased from the State Key Laboratory of Molecular

Oncology, Chinese Academy of Medical Sciences (Beijing, China).

Expression and Puriﬁcation of DCN1−5 Proteins. Human

pDEST17-DCN1 (residues 58−259) plasmid containing N-terminal

His₆tag was a gift from Dr. Shaomeng Wang’s group at the University

of Michigan, Ann Arbor. DCN1 (residues 58−259), DCN2 (residues

62−259), DCN3 (residues 86−304), DCN4 (residues 102−292),

and DCN5 (residues 47−237) were cloned into an N-terminal GST

tag plasmid pGEX4T-1. The plasmids pDEST17-DCN1 and

pGEX4T-1-DCN1−5 were transformed into E. coli BL21-AI and

BL21(DE3), respectively. A preculture was grown in medium

containing 50 μg/mL ampicillin at 37 °C. When the cells grew

until OD₆₀₀> 0.7 after about 5−8 h, 4 mg/mL L-(+)-Arabinose and

0.25 mM isopropyl-β-^D-galactopyranoside (IPTG) were added to the

culture medium to induce the protein synthesis for 8 h at 20 °C.

Subsequently, the cells were collected and disrupted in ice-cold

washing buﬀer (25 mM Tris-HCl pH 7.5, 1 mM DTT, 200 mM

4-(3-Chlorophenyl)-6-((1-(2-(dimethylamino)ethyl)-1H-tetrazol-

5-yl)thio)-2-(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (47).

1

Yield 75.2%. Yellow solid. Mp: 179−180 °C. H NMR (400 MHz,

DMSO-d₆, ppm): δ 8.02 (s, 1H, Ar−H), 7.97 (d, J = 7.9 Hz, 1H, Ar−

H), 7.81−7.75 (m, 1H, −CH), 7.69 (t, J = 7.9 Hz, 1H, −CH),

4.99 (t, J = 6.6 Hz, 2H, −CH₂−), 3.69 (t, J = 5.1 Hz, 2H, −CH₂−),

3.67 (d, J = 2.2 Hz, 2H, −CH₂−), 3.21 (t, J = 2.4 Hz, 1H, C−H),

2.84 (s, 6H, −CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ

172.34, 169.87, 165.06, 146.17, 136.11, 133.61, 132.04, 130.93,

128.64, 127.64, 114.45, 99.20, 78.85, 73.69, 54.14, 42.72, 42.41, 19.24.

HR−MS (ESI) calcd for C₁₉H₁₈ClN₈S₂: [M + H]⁺m/z, 457.0784;

found, 457.0785.

4-((1-(2-(Dimethylamino)ethyl)-1H-tetrazol-5-yl)thio)-2-(prop-2-

yn-1-ylthio)-6-(p-tolyl)pyrimidine-5-carbonitrile (48). Yield 46.0%.

White solid. Mp: 190−191 °C. ¹H NMR (400 MHz, DMSO-d₆,

ppm): δ 7.94 (d, J = 8.2 Hz, 2H, Ar−H), 7.45 (d, J = 8.2 Hz, 2H, Ar−

H), 4.97 (t, J = 6.4 Hz, 2H, −CH₂−), 3.65 (d, J = 2.3 Hz, 4H,

−CH₂−), 3.20 (t, J = 2.3 Hz, 1H, C−H), 2.81 (s, 6H, −CH₃), 2.43

(s, 3H, −CH₃). ¹³C NMR (100 MHz, DMSO-d₆, ppm): δ 172.05,

169.89, 166.23, 146.24, 142.93, 131.32, 129.54, 129.01, 114.87, 98.23,

78.92, 73.67, 54.29, 42.83, 42.49, 21.11, 19.13. HR−MS (ESI) calcd

for C₂₀H₂₁N₈S₂: [M + H]⁺m/z, 437.1331; found, 437.1330.

4-(3,4-Dichlorophenyl)-6-((1-(2-(dimethylamino)ethyl)-1H-tetra-

zol-5-yl)thio)-2-(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile (49).

1

Yield 46.0%. White solid. Mp: 174−175 °C. H NMR (400 MHz,

DMSO-d₆, ppm): δ 8.23 (s, 1H, Ar−H), 7.97 (dd, J = 17.7, 8.2 Hz,

2H, Ar−H), 4.98 (t, J = 6.3 Hz, 2H, −CH₂−), 3.69 (s, 2H, −CH₂−),

3.68 (s, 2H, −CH₂−), 3.21 (s, 1H, C−H), 2.83 (s, 3H, −CH₃). ¹³

C

NMR (100 MHz, DMSO-d₆, ppm): δ 172.40, 169.87, 164.10, 146.12,

135.29, 134.53, 131.88, 131.33, 130.77, 129.03, 114.35, 99.21, 78.82,

73.70, 54.15, 42.74, 42.44, 19.27. HR-MS (ESI) calcd for

C₁₉H₁₇C_l2N₈S₂: [M + H]⁺m/z, 491.0395; found, 491.0395.

4-((1-(2-(Dimethylamino)ethyl)-1H-tetrazol-5-yl)thio)-6-(2-oxo-

2H-chromen-6-yl)-2-(prop-2-yn-1-ylthio)pyrimidine-5-carbonitrile

1

(50). Yield 72.4%. Yellow solid. Mp: 197−198 °C. H NMR (400

MHz, DMSO-d₆, ppm): δ 8.39 (s, 1H, Ar−H), 8.27−8.19 (m, 2H,

Ar−H), 7.66 (d, J = 8.7 Hz, 1H, Ar−H), 6.63 (d, J = 9.6 Hz, 1H, Ar−

H), 5.00 (t, J = 6.5 Hz, 2H, −CH₂−), 3.69 (d, J = 4.9 Hz, 2H,

−CH₂−), 3.66 (d, J = 1.9 Hz, 2H, −CH₂−), 3.23 (t, J = 2.3 Hz, 1H,

C−H), 2.83 (s, 6H, −CH₃). ¹³C NMR (100 MHz, DMSO-d₆,

ppm) δ 172.25, 169.97, 165.07, 159.32, 155.78, 146.19, 143.74,

132.17, 130.26, 129.65, 119.12, 117.43, 117.17, 114.64, 98.72, 78.82,

73.79, 54.23, 42.81, 42.46, 42.34, 19.22. HR−MS (ESI) calcd for

C₂₂H₁₈N₈NaO₂S₂: [M + Na]⁺m/z, 513.0892; found, 513.0893.

2-(Allylthio)-4-(4-chlorophenyl)-6-(thiazol-2-ylthio)pyrimidine-5-

1

carbonitrile (51). Yield 56.0%. White solid. Mp: 109−110 °C. H

NMR (400 MHz, CDCl₃): δ 8.19 (d, J = 3.3 Hz, 1H, Ar−H), 8.11 (d,

J = 3.3 Hz, 1H, Ar−H), 8.03−7.85 (m, 2H, Ar−H), 7.77−7.60 (m,

2H, Ar−H), 5.70 (dd, J = 16.9, 10.1 Hz, 1H, −CH), 5.28−4.98 (m,

2H, −CH₂−), 3.56 (d, J = 6.8 Hz, 2H, −CH₂−). ¹³C NMR (101

MHz, DMSO): δ 173.13, 171.37, 165.31, 150.35, 144.44, 137.05,

133.23, 132.71, 130.83, 128.94, 128.09, 118.36, 114.57, 97.88, 33.16.

HR−MS (ESI) calcd for C₁₇H₁₁ClN₄NaS₃: [M + Na]⁺m/z, 424.9732;

found, 424.9730.

4-(4-Chlorophenyl)-2-(propylthio)-6-(thiazol-2-ylthio)-

pyrimidine-5-carbonitrile (52). Yield 48.2%. White solid. Mp: 112−

113 °C. ¹H NMR (400 MHz, DMSO): δ 8.25 (d, J = 3.3 Hz, 1H, Ar−

H), 8.15 (d, J = 3.3 Hz, 1H, Ar−H), 8.02−7.89 (m, 2H, Ar−H), 7.70

(dd, J = 8.9, 2.1 Hz, 2H, Ar−H), 2.82 (t, J = 7.2 Hz, 2H, −CH₂−),

1.46 (dd, J = 14.5, 7.3 Hz, 2H, −CH₂−), 0.86 (t, J = 7.3 Hz, 3H,

−CH₃). ¹³C NMR (101 MHz, DMSO): δ 173.90, 171.38, 165.19,

150.25, 144.56, 136.97, 133.29, 130.79, 128.93, 128.26, 114.65, 97.58,

Q

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NaCl) by sonication. The soluble and pellet fractions were separated

by centrifugation at 12 000g for 15 min. After ﬁltration, cleared ﬁltrate

containing N-terminal His₆tag protein was applied to a Ni-beads

column and washed with washing buﬀer containing diﬀerent

concentration of imidazole. The ﬁltrate containing N-terminal GST

tag protein was then puriﬁed by GST-trap column and washed with

washing buﬀer containing 10 mM glutathione. Afterward, the

molecular weights of these proteins were conﬁrmed by Commassie

Blue staining, and the concentration of the recombinant proteins was

determined, using a Bicinchoninic Acid (BCA) Protein Assay kit.

Competitive FP Binding Assay. The ﬂuorescence polarization

(FP) competitive binding assays were performed similarly as

described previously.^30,32FP assays were carried out in 96-well,

black, round-bottom microtiter plates. The FAM-labeled ﬂuorescent

probe (FAM-782) with a K_dvalue of 120.43 nM for DCN1 and the

analyzing protocol³⁵were kindly provided by Shaomeng Wang’s

group at the University of Michigan, Ann Arbor for compound

screening.³⁰DCN1 containing N-terminal His₆tag was used for FP

binding assay. DCN1 and the probe were mixed to a ﬁnal volume of

100 μL in the assay buﬀer (100 mM phosphate buﬀer, pH = 6.5,

0.02% Tween-20). After being shaken at room temperature for 30

min, the plates were measured by PerkinElmer Envision microplate

reader at an excitation wavelength of 485 nm and an emission

wavelength of 530 nm. The data were analyzed by SPSS 20 and

GraphPad Prism 5 software. K_ivalues were calculated on the basis of

the methods described earlier.^32,74The equation is K_i= [I]₅₀/([L]₅₀/

K_d+ [P]₀/K_d+ 1), where [I]₅₀is the concentration of the inhibitor at

50% inhibition, [L]₅₀is the concentration of the ligand at 50%

inhibition, K_dis the dissociation constant of the protein−ligand

complex, and [P]₀is the concentration of the protein at 0% inhibition.

HTRF Assay. HTRF assays were carried out in 384-well, white,

round-bottom microtiter plates at a ﬁnal volume of 20 μL per well.

The assay cocktail was prepared as a mixture of HTRF detection

buﬀer (62SDBRDD), Streptavidin-XL665 (610SAXLA), Anti-GST-

Cryptate Gold (61GSTKLA), 5 nM GST-DCN1−5, 5 nM

AcUBE2M¹⁻²¹-biotin, in assay buﬀer (100 mM phosphate buﬀer,

pH = 6.5, 0.02% Tween-20). The AcUBE2M¹⁻²¹-biotin peptide was

synthesized by China Peptide Co. (Shanghai, China), and its

sequence is Ac-MIKLFSLKQQKKEEESAGGTK-biotin. The cocktail

was incubated for 30 min at room temperature. Compounds to be

screened were added to assay plates, and the control well was added

in the same volume of DMSO solution. The assay mixture was

incubated for 30 min at room temperature before the measurement of

the HTRF signal with a PerkinElmer Envision microplate reader

equipped with modules for excitation at 320 nm and emission at 615

and 665 nm. The data were analyzed by SPSS 20 and GraphPad

Prism 5 software. K_ivalues were calculated on the basis of the

methods described earlier.⁷⁷

Molecular Docking. The crystal structure (PDB ID: 3TDU,

resolution of 1.5 Å) of human Cul1WHB-Dcn1P-acetylated Ubc12N

complex was obtained from the RCSB protein data bank. The

preparation of protein structure was performed under the Amber 10:

EHT force ﬁeld, using the Quickprep module with the default

parameters, which included the addition of hydrogen atoms, the

deletion of solvent, the repair of the missing residues, and the setup of

the protonation states of the ionizable residues with pK_a= 7. The

ligand was prepared under Amber 10: EHT force ﬁeld by energy

minimization and conformational search. Afterward, we got the

conformational database of the ligand structure for further docking

study. All of the conformations then were docked into the binding

pocket of human DCN1 with the default parameters, using the

method of molecular docking. The docking structure was scored by

GBVI/WSA dG and held 20 docking poses in the case of parameters

by default.

Coimmunoprecipitation Assay. H1975 cells were treated with

1‰ DMSO, 10 μM compound DC-2N, DC-2, or NAcM-COV,

respectively, for 5 h. The cells then were collected and lysed with

RIPA buﬀer. The whole cell lysate was incubated by UBE2M antibody

and protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotech, CA).

DCN1 and UBE2M protein associated with beads was eluted by

heating and detected by immunoblotting.

Cell-Free NAE Activity Assay. NAE activity assay (in vitro

NEDD8∼UBE2M formation assay) was conducted according to the

manufacturer’s instructions. In brief, NEDD8 E1 was mixed with

NEDD8, UBE2M, and 10 and 20 μM MLN4924 or 1, 10, and 100

μM DC-2 in the reaction buﬀer and incubated at room temperature

for 10 min. The reaction was initiated by the addition of Mg-ATP

solution. After 60 min of incubation at 37 °C, the reaction mixtures

were added with 1 mM DTT and protein loading buﬀer (no DTT).

The mixtures were then separated by a 12%SDS-PAGE gel.⁷⁶

Cell Viability Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-

trazolium bromide (MTT) was used to measure the cell viability,

according to the manufacturer’s instructions. After the cells were

seeded into 96-well plates at a concentration of (2−5) × 10³cells per

well, serial dilutions of compounds were added and cultured for 72 h.

Next, MTT solution (5 mg/mL in PBS) was added and incubated at

37 °C for 4 h. After 150 μL of DMSO was added followed by

removing the MTT, the plate was measured at the absorbance of 490

nm. The data were analyzed using SPSS 20 software.

Colony Formation Assay. First, H1975, PC9, and A549 cells

were seeded at a concentration of 1 × 10³cells per well and cultured

overnight. The fresh medium then was added with compound DC-2

into the wells. After 7 days of incubation, the cells were ﬁxed and

stained by 0.1% crystal violet solution and then imaged by microscopy

(Nikon). Subsequently, the crystal violet crystals were dissolved and

measured by a BioTek microplate reader. The data were analyzed

using the GraphPad Prism 5 software.

Normal Thermal Shift Assay. The assay was performed on a

C1000 Touch Thermal cycler-CFX96TM Real-Time PCR (BIO-

RAD), using the 96-well thin-wall PCR plate. The buﬀer used in this

experiment is 100 mM phosphate buﬀer, pH = 6.5, 0.02% Tween-20.

A total volume of 25 μL of solution containing 0.2 mg/mL DCN1

protein, compounds (DC-2, DC-2N, and NAcM-COV), and

1×Sypro Orange dye was dispensed into the 96-well plate. The

same volume of DMSO was used as control. The plates then were

heated in a cycler from 25 to 95 °C (71 heating cycles in 35.5 min).

The data were analyzed by BIO-RAD software.⁵⁶

Analysis of Cellular Apoptosis. For the analysis of apoptosis by

ﬂow cytometry, ﬁrst, H1975, PC9, and A549 cells were seeded into 6-

well plates ((1−2) × 10⁶cells/mL) and incubated overnight.

Compound DC-2 at 0, 2.5, 5, and 10 μM was then added. After

treatment for 48 h, the cells were harvested and stained using the

Annexin-V-FITC/PI apoptosis kit for 30 min. The cells were then

collected and measured by a FACSCalibur ﬂow cytometer (BD

Biosciences). All data were analyzed by FlowJo-V10 and GraphPad

Prism 5 software.

Cellular Thermal Shift Assay (CETSA). CETSA was performed

to detect the ability of compounds interacting with its targets at

cellular level.⁷⁵First, the cultured cells were treated with cell media

containing 1‰ DMSO or 10 μM compounds DI-591, DC-2, and

DC-2N for 3−5 h, respectively, the cells were collected, aliquoted into

seven PCR tubes, and then heated for 10 min from 43 to 61 °C. As

for the dose-dependent assay, 1‰ DMSO, 0.3, 1, 3, 10 μM DC-2, or

10 μM DC-2N was added into the cells for 3−5 h and then heated for

10 min at 55 °C. After that, the cells were lysed by three repeated

cycles of freeze−thawing, using liquid nitrogen. Finally, after being

centrifuged at 12 000g for 20 min, the supernates were used for

Western Blot analysis.

Western Blot Analysis. H1975 and PC9 cells were seeded in 100

mm²plastic dishes (1 × 10⁶cells/well) and incubated overnight prior

to the addition of compound DC-2 at 0, 0.3, 1, 10, and 20 μM for 24

h. The cells then were harvested, lysed, and centrifuged at 12 000g for

20 min at 4 °C. The supernatant was determined by a BCA Protein

Assay kit, denatured at 100 °C for 10 min, and incubated at −20 °C

for Western Blot. After being separated by SDS-PAGE, the protein

was transferred to 0.22 μm nitrocellulose membranes and blocked by

5% skim milk. The membranes then were probed with appropriate

primary antibodies and incubated at 4 °C overnight, followed by the

treatment of horseradish-peroxidase-conjugated secondary antibody

(1:10 000). Subsequently, the membranes were washed with PBST

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DOI: 10.1021/acs.jmedchem.9b00003

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

and examined by enhanced chemiluminescence. The data were

determined and analyzed using ImageJ software and GraphPad Prism

5 software.

Notes

The authors declare no competing ﬁnancial interest.

Water Solubility Assay. Water solubility assay was performed as

previous described.^30,73Brieﬂy, compound DC-2 was added as

powder into an Eppendorf tube, which was full of phosphate buﬀer

(PBS, pH 7.4), until a heterogeneous suspension was obtained. The

suspension then was sonicated in a water bath for 30 min and shaken

for 24 h at room temperature until reaching thermodynamic

equilibrium. Suspensions were then centrifuged at 12 000g for 15

min, and the supernatants were ﬁltered, using 0.45 μm membrane.

PBS was used as a control. The ﬁltrate was measured by UV-2700

spectroscopy (Shimadzu, Japan).

ACKNOWLEDGMENTS

■

We thank Dr. Brenda A. Schulman (Department of Structural

Biology and Howard Hughes Medical Institute, St. Jude

Children’s Research Hospital, Memphis, TN) and Dr. R. Kip

Guy’s group (Department of Pharmaceutical Sciences,

University of Kentucky, Lexington, KY) for providing the

compounds NAcM-COV, NAcM-OPT, and NAcM-NEG.

This work was supported by a National Key Grant from the

Chinese Ministry of Science and Technology

(2016YFA0501800 to W.Z.), the National Natural Science

Foundation of China (nos. 81470524 and 81870297 to W.Z.;

no. 81430085 to H.-M.L.; and no. 81703328 to L.M.), and the

Henan Scientiﬁc Innovation Talent Team, Department of

Education (19IRTSTHN001 to W.Z.); the Starting Grant of

Zhengzhou University (Grant 32210535 to L.M.); and the

Scientiﬁc Program of Henan Province (Grant 182102310070

to L.M.).

siRNA Experiments. Cells were seeded in six-well plates for 16−

24 h before siRNA treatment. Medium then was taken away, and the

cells were transfected with siRNA using Lipofectamine RNAiMAX

Reagent according to the manufacturer’s instructions. Three days

later, the cells were collected and used for Western Blot assay. Three

diﬀerent siRNA duplexes targeting three speciﬁc sequences of DCN1

and a negative control siRNA were synthesized by GenePharma

(Shanghai, China). The sense strand nucleotide sequence for DCN1

#1 siRNA was 5′-GGAUAAAGUUCGUCAGUUUTT-3′, and the

antisense sequence was 5′-AAACUGACGAACUUUAUCCTT-3′;

DCN1 #2 siRNA was 5′-GGACAGGAAGAAGUUAGAATT-3′,

and the antisense sequence was 5′-UUCUAACUUCUUCCU-

GUCCTT-3′; DCN1 #3 siRNA was 5′-GCCAUUGCCUACUG-

GAACUTT-3′, and the antisense sequence was 5′-AGUUCCA-

GUAGGCAAUGGCTT-3′. The negative control siRNA (siNeg)

sense sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′, while

the antisense sequence was 5′-ACGUGACACGUUCGGAGAATT-

3′.

ABBREVIATIONS USED

■

DCN1, defective in cullin neddylation 1; UBE2M, NEDD8-

conjugating enzyme Ubc12; SCC, squamous cell carcinoma;

CRL, cullin ring ligases; NEDD8, neural precursor cell

expressed, developmentally down-regulated 8; NAE,

NEDD8-activating enzyme; RBX1, ring-box 1; E3, ubiquitin

protein ligase; CUL1, cullin 1; CUL2, cullin2; CUL3, cullin3;

CUL4, cullin4; CUL5, cullin5; MTT, 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide; CETSA, cellular thermal

shift assay; FP, ﬂuorescence polarization; IPTG, isopropyl-β-^D-

galactopyranoside; HTRF, homogeneous time-resolved ﬂuo-

rescence; NRF2, the nuclear factor-erythroid 2 related factor 2;

SAR, structure−activity relationship; NQO1, NADPH:

quinone oxidoreductase-1; HO-1, heme oxygenase-1

Statistical Analysis. Data were expressed as mean

SD.

Statistical diﬀerences in two groups were performed by student’s t-

test using GraphPad Prism 5 software. One-way ANOVA analysis was

used for multiple group comparison by SPSS 20 software. The P-

values of 0.05 or less were considered statistically signiﬁcant.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.9b00003.

■

S

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AUTHOR INFORMATION

Corresponding Authors

■

(6) Zhong, H. J.; Leung, K. H.; Lin, S.; Chan, D. S.; Han, Q. B.;

Chan, S. L.; Ma, D. L.; Leung, C. H. Discovery of deoxyvasicinone

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*Phone: 86-371-67781739. E-mail: liuhm@zzu.edu.cn.

*Phone: 47-98280093. E-mail: zhenhe.suo@medisin.uio.no.

*Phone: 86-15003898187. E-mail: zhaowen100@139.com,

zhaowen@zzu.edu.cn.

ORCID

Wenjuan Zhou: 0000-0001-8151-1125

Shaomeng Wang: 0000-0002-8782-6950

Hong-Min Liu: 0000-0001-6771-9421

Wen Zhao: 0000-0002-2530-7637

Author Contributions

^∥W.Z. and L.M. contributed equally to this work.

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Potent 5-Cyano-6-phenyl-pyrimidin-Based Derivatives Targeting DCN1-UBE2M Interaction

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